Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-[1-¹⁴C]arachidonoyl PC or 1-acyl-2-[1-¹⁴C]linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-[1-¹⁴C]linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label being transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-[1-¹⁴C]arachidonoyl PC released¹⁴C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca²⁺-ionophore A₂₃₁₈₇. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.     

Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5°C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual subtrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.     

A highly active soluble diacylglycerol synthesizing system from developing rapeseed,Brassica napus L.

The subcellular distribution of the enzymes of triacylglycerol biosynthesis has been studied in developing oilseed rape. All in vitro enzymatic activities from oleoyl-CoA to triacylglycerol were sufficient to account for the known rate of oleate deposition in triacylglycerol in vivo. The enzymatic activities from oleoyl-CoA to diacylglycerol preferentially were localized in a 150,000 g supernatant fraction, while the diacylglycerol acyl-transferase mostly was associated with the microsomal (20,000 g pellet and 150,000 g pellet) and oil-body fractions. The soluble (150,000 g supernantant) fraction rapidly incorporated oleate from [1-¹⁴C]oleoyl-CoA into diacylglycerol with rates of 40 nm min⁻¹ g⁻¹ FW at 20 μM oleoyl-CoA. The pH optimum was 7.5–9.0, and normal saturation kinetics were seen with oleoyl-CoA; the S_0.5 was about 32 μM. Exogenous acyl acceptors, such as glycerol 3-phosphate, lysophosphatidic acid and lysophosphatidylcholine stimulated oleate incorporation into diacylglycerol. The detergents Triton X-100 and sodium cholate inhibited diacylglycerol formation at concentrations in the region of their critical micellar concentration, while n-octyl-β,D-glyco-pyranoside had no effect, even at high concentration. The significance of these findings for the mechanism of oil-body formation in developing oilseeds is discussed.     

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[¹⁴C]rici-noleoyl-sn-glycero-3-phosphocholine, ¹⁴C-labeled 2-linoleoyl-phosphatidylcholine (PC), and ¹⁴C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A₂ strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[¹⁴C]ricinoleoyl-PC and [¹⁴C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC. Out results demonstrate the proposed pathway: 2-oleoyl-PC→2-ricinoleoyl-PC→ricinoleate →triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.     

Properties of lysophosphatidylcholine acyltransferase from <Emphasis Type="Italic">Brassica napus</Emphasis> cultures

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce PC and CoA. LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed TAG composition. LPCAT activity in microsomes prepared from microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf) was assayed using [1-¹⁴C]acyl-CoA as the fatty acyl donor. LPCAT activity was optimal at neutral pH and 35°C, and was inhibited by 50% at a BSA concentration of 3 mg mL⁻¹. At acyl-CoA concentrations above 20 μM, LPCAT activity was more specific for oleoyl (18∶1)-CoA than stearoyl (18∶0)- and palmitoyl (16∶0)-CoA. Lauroyl (12∶0)-CoA, however, was not an effective acyl donor. LPC species containing 12∶0, 16∶0, 18∶0, or 18∶1 as the fatty acyl moiety all served as effective acyl acceptors for LPCAT, although 12∶0-LPC was somewhat less effective as a substrate at lower concentrations. The failure of LPCAT to catalyze the incorporation of a 12∶0 moiety from acyl-CoA into PC is consistent with the tendency of acyltransferases to discriminate against incorporation of this fatty acyl moiety at the sn-2 position of TAG from the seed oil of transgenic B. napus expressing a medium-chain thioesterase.     

Effects of sodium butyrate on the transfer of arachidonic acid to phosphatidylcholine in a clonal oligodendrocyte cell line (CB-II)

The effect of sodium butyrate on membrane phospholipid metabolism in a neonate rat cerebellum derived clonal oligodendrocyte cell line (CB-II) was investigated. Sodium butyrate is an agent known to induce cell differentiation and morphological transformations. A comparison of thein vivo phospholipid labeling patterns obtained by incubating CB-II cells with [³H]choline, [¹⁴C]myristic acid or [³H]arachidonic acid indicated that butyrate altered the route of acylation-deacylation in phosphatidylcholine (PC) biosynthesis. Using anin vitro incubation system containing homogenates of CB-II cells, the largest proportion of radioactivity was found in PC, and addition of sodium butyrate resulted in a further increase in the transfer of arachidonic acid to PC, but not to phosphatidylinositol. Similar results were obtained when thisin vitro acylation activity was tested using homogenates from sodium butyrate pretreated cells. The butyrate effect was observed regardless of whether or not exogenous lysophosphatidylcholine (LPC) was added to the incubation system. Addition of butyrate did not result in a change in the activity of LPC:acyl-CoA (coenzyme A) acyltransferase (EC 2.3.1.23) in CB-II cells upon incubating cell homogenates with [1-¹⁴C]arachidonoyl-CoA and LPC. However, when cell homogenates were incubated with [³H]arachidonic acid in the presence of 2.5–10 mM sodium butyrate, arachidonoyl-CoA synthesis was stimulated. A time course study demonstrated that significant stimulation occurred after three minutes. Taken together, the results suggest that in CB-II cells, sodium butyrate stimulates the transfer of arachidonic acid into PC and that this effect is at least partially due to a stimulation of arachidonoyl-CoA ligase (EC 6.2.1.3).     

The effect of protein deficiency on some rat liver lipid metabolic enzymes and CoA

Two groups of male Wistar rats were fed normal (i.e., 18%) and protein-free diets, respectively, for 7 weeks. In vivo incorporation of [1-¹⁴C] acetate into palmitic, stearic, oleic, and arachidonic acids by the liver was reduced in the protein-deficient rats. In vitro incubation of liver microsomes with labeled palmitate or linoleate revealed no change in the specific activities of chain elongating or desaturating enzymes. Protein deficiency resulted in a decrease in specific activity of short chain acyl-CoA synthetase and in total CoA, accompanied by the virtual disappearance of acyl-CoA and an increase in free CoA. Furthermore, there was less microsomal fatty acid synthetase and mitochondrial β-hydroxybutyrate dehydrogenase activity. These results are discussed in relation to fatty acid synthesis and the changes in liver fatty acid composition.     

Metabolism of n−3 polyunsaturated fatty acids by the isolated perfused rat liver

The hepatic metabolism of oleic acid and n−3 fatty acids (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and secretion of very low density lipoprotein (VLDL) were studied in isolated perfused rat livers from normal chow fed male rats. The basal perfusion medium contained 30% bovine erythrocytes, 6% bovine serum albumin (BSA), and 100 mg/dL glucose, in Krebs-Henseleit bicarbonate buffer (pH 7.4) which was recycled through the liver for 2 hr. Individual fatty acids (EPA, DHA or oleic acid), as complexes with 6% BSA, or albumin alone, were infused at a rate of 70 μmol/hr. When any of these fatty acids was infused at this rate, the ambient concentration in the medium was maintained at 0.3–0.4 μmol/mL, indicative of similar hepatic rates of uptake for each fatty acid (i.e., approximately 6 μmol/g liver/hr). When fatty acid was not infused, the ambient free fatty acid level was 0.16 μmol/mL. The concentrations of infused free fatty acids increased appropriately in the perfusion medium; however, with infusion of EPA, DHA, or oleate, the concentrations of perfusate palmitate and linoleate were the same as when fatty acid was not infused. Additionally, the perfusate concentration of oleate in the free fatty acid fraction was not affected by infusion of EPA and DHA. These data indicate a constant outflow of endogenous fatty acid unaffected by the presence of the exogenously supplied fatty acid. The net secretion rate of VLDL lipids and protein was stimulated by infusion of oleate, whereas when EPA was infused, secretion rates were lower and similar [except for VLDL cholesterol (C), which was greater] to those occuring when fatty acid was not provided. DHA stimulated the secretion of VLDL triacylglycerol (TG), phospholipid (PL) and C to a similar rate, as did oleate, but secretion of VLDL cholesteryl ester (CE) and protein was lower and similar to that with EPA. VLDL and hepatic TG and CE were enriched with the infused fatty acids, compared to experiments without fatty acids, as determined by gas chromatography. Enrichment of PL, however, was significant only in liver upon infusion of EPA. The formation of¹⁴CO₂ and perchloric acid soluble products from [1-¹⁴C]EPA, considered separately, did not differ statistically from that obtained with [1-¹⁴C]oleate, although the mean values were higher with [1-¹⁴C]EPA. However, the sum of oxidation products derived from EPA was significantly greater than that from oleate. Incorporation of [1-¹⁴C]EPA into TG and CE, but not into PL, was lower as compared to that from [1-¹⁴C]oleate. These lower rates of incorporation of [1-¹⁴C]EPA into VLDL lipids therefore paralleled the mass fatty acid enrichment-patterns. It may be concluded that EPA is used to a similar extent as oleate for synthesis of PL, but is a poorer substrate for synthesis of TG. The reduced output of newly synthesized (radioactive) PL reflected the lower hepatic output of VLDL. Since hepatic uptake of EPA, DHA or oleate was identical, utilization of EPA for TG synthesis was less than that of oleate or DHA. Further-more, utilization of endogenous fatty acids for TG synthesis and secretion of the VLDL was reduced in the presence of EPA. The decreased TG synthesis resulted in reduced formation of VLDL for transport of TG from the liver. These effects taken together with an apparently increased oxidation of EPA provide substantial evidence for a decrease in formation of VLDL and transport of TG, PL, C and CE into the circulation in response to EPA. DHA, however, appears to be an adequate substrate for TG synthesis and stimulates VLDL secretion. The reduced transport of CE may reflect lower selectivity of DHA by acyl-CoA; cholesterol acyltransferase for CE formation.     

One-step synthesis of radioactive acyl-CoA and acylcarnitines using rat liver mitochondrial outer membrane as enzyme source

Rat liver mitochondrial outer membrane enriched preparations have proven to be a convenient enzyme source for synthesizing coenzyme A (CoA) and carnitine esters of radioactive fatty acids. These membranes are simple to isolate and they retain acyl-CoA ligase and carnitine palmitoyltransferase activities well upon storage. Enzyme purification is not required. A novel aspect of the present procedure is that the same enzymatic incubation step allows both the acyl-CoA and the acylcarnitine esters to be obtained simulataneously when carnitine is present, but produces acyl-CoA ester only when carnitine is not included. Under the conditions described, the conversion of [1-¹⁴C]octanoic acid to the respective esters was about 95%; the corresponding figure for [1-¹⁴C]palmitic acids was over 70%. The procedure seems suitable for synthesizing the labeled CoA and carnitine esters from a variety of radioactive fatty acids. A preliminary account of this work has been published (ref. 1).     

Saturated fatty acids >C20 are not activated by acid:CoA ligase in rat brain or liver?

The formation of long-chain saturated acyl-[³H]CoA and [1-¹⁴C] acyl-[³H]CoA by rat brain microsomes and rat liver was examined. Acyl-CoA formation was markedly decreased as fatty-acid chain length increased from C₁₆ to C₂₀. No biosynthesis of behenyl-[³H] CoA or [1-¹⁴C] lignoceryl-[³H] CoA was observed. The results suggest that long-chain saturated fatty acids >20 carbons in length are not activated by acid:CoA ligase to form acyl-CoA.     

Effect of Δ9-tetrahydrocannabinol and merthiolate on acyltransferase activities in guinea pig liver microsomesand merthiolate on acyltransferase activities in guinea pig liver microsomes

Δ⁹-tetrahydrocannabinol (THC) and merthiolate have been utilized as lysophospholipid acyltransferase inhibitors in metabolic studies. However, their effects on acyltransferases other than lysophosphatidylcholine:acyl-CoA acyltransferase (LPCAT) are not known. We have therefore investigated the effectiveness of THC and merthiolate in inhibiting the acylation of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol (LPI) and lysophosphatidic acid (LPA) in guinea pig liver microsomes using oleoyl-CoA and arachidonoyl-CoA as acyl donors. THC inhibited LPCAT and lysophosphatidylethanolamine: acyl-CoA acyltransferase (LPEAT) by 40–50%, but had no effect or only slightly increased the activities of the other acyltransferases when assayed with oleoyl-CoA as the acyl donor. The results obtained with arachidonoyl-CoA were similar to those with oleoyl-CoA, with the exception of a 40% inhibition of lysophosphatidylserine:acyl-CoA acyltransferase (LPSAT) at concentrations of 50 μM or higher. At similar concentrations, merthiolate was more effective than THC in inhibiting the acyltransferases examined. Selective effects on the acyltransferases were observed at low concentrations of merthiolate (20 μM or less). Thus, LPCAT was most susceptible, followed by LPI acyltransferases, LPSAT, LPEAT and lysophosphatidic acid:acyl-CoA acyltransferases (LPAAT). The presence of LPA did not affect the inhibition of LPCAT by merthiolate. Thus the resilience of LPAAT to merthiolate inhibition was not due to chelation of the compound by the acidic lysolipid. Thiol reagents includingN-ethylmaleiamide, 5,5′-dithio-bis-nitrobenzoic acid, iodoacetate, β-mercaptoethanol and dithiothreitol had little or no effect on the acyltransferases relative to equimolar concentrations of merthiolate. The above results indicate that merthiolate is a much more effective inhibitor of lysophospholipid:acyl-CoA acyltransferases than is THC, and that the selectivity exhibited by merthiolate may be due to direct and specific interaction with the acyltransferases.     

Acetate and mevalonate labeling studies with developingCuphea lutea seeds

Cuphea seeds contain large amounts of medium chain (C₈ to C₁₄) fatty acids, mainly as triacylglycerols. The biosynthesis of these lipids was studied in vivo by incubating developingCuphea lutea seeds with labeled acetate. Incorporation of label into triacylglycerols and into medium chain fatty acids occurred principally during the period of endogenous lipid deposition, but some label was encountered in these products even during seed dehydration. At this later stage palmitate and oleate were the dominant labeled fatty acids. During the period of rapid endogenous lipid deposition acyl lipids other than triacylglycerols were minor labeled components. The labeling patterns were consistent with the Kennedy pathway for triacylglycerol biosynthesis. The fatty acid composition of the acyl-CoA pool was similar to the total lipid fatty acid composition, but the acyl-ACP pool contained relatively more short chain acyl groups. Squalene was labeled from acetate throughout the period of seed development, but labeled sterols were not detected. Using [2-¹⁴C]mevalonic acid lactone as substrate, squalene was the principal labeled product. Small amounts of label were found in free sterols. However, in terms of mass, free sterol dominated over squalene. The possibility of two independent sites of isoprenoid biosynthesis in the developing embryo is discussed.     

Substrate specificity of lysosomal cholesteryl ester hydrolase isolated from rat liver

The effect of various physicochemical forms of substrate on the activity of acid cholesteryl ester hydrolase isolated from rat liver lysosomes was studied. The amount of sodium taurocholate was varied in the substrate mixture which contained constant amounts of egg phosphatidylcholine (PC) and cholesteryl oleate. The resulting substrate forms produced were PC vesicles, PC vesicles with incorporated sodium taurocholate, mixed micelles, and mixed micelles together with free bile salt micelles. Gradually increasing amounts of sodium taurocholate activated cholesteryl oleate hydrolysis until the molar sodium taurocholate/PC ratio of ca. 0.6; thereafter hydrolytic activity decreased rapidly. The presence of sodium taurocholate micelles clearly inhibits cholesteryl oleate hydrolysis. We therefore propose that the activation observed at low bile salt concentrations depends on bile salt interaction with the substrate vehicle, whereas the inhibition observed at high bile salt concentrations depends on sodium taurocholate interacting with the enzyme. When comparing different phospholipid components in the supersubstrate, the enzyme activity was highest in the presence of dioleyl PC and decreased when present with dipalmitoyl PC and egg PC. Egg lysoPC completely inhibited the enzyme activity. A net negative charge on the surface of the vesicle substrate increased cholesteryl ester hydrolase activity while a net positive charge on the surface inhibited the enzyme activity. Only part of the product inhibition of cholesteryl oleate hydrolase caused by Na-oleate was reversible when tested with bovine serum albumin present in the incubation mixture.     

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol systhetase of rat intestinal mucosa. With 2-oleoyl[-³H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97–98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was thesn-1,2-and 10% thesn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was thesn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-³H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.     

Specific formation of arachidonoyl phosphatidylinositol from 1-acyl-sn-glycero-3-phosphorylinositol in rat liver

The conversion of 1-acyl-sn-glycero-3-phosphorylinositol-³H into phosphatidylinositol-³H was studied using rat liver microsomal and homogenate preparations. The nature of the molecular species of phosphatidyl inositol so formed in the absence of added acyl moieties was determined after fractionating the radioactive product by means of argentation thin layer chromatography. In other experiments, the possible specificity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase towards different acyl-CoA derivatives was investigated. Maximum conversion of 1-acyl GPI to the diacyl analogue was dependent on the addition of adenosine triphosphate and CoA when exogenous acyl groups were omitted from the incubation medium. Under these latter conditions, the tetraenoic species comprised 56–74% of the total molecular species of newly-formed phosphatidylinositol. The microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphorylinositol acyltransferase showed a marked preference for arachidonoyl-CoA. The present results suggest that the enrichment of rat liver phosphatidyl inositol in arachidonic acid may arise when 1-acyl-sn-glycero-3-phosphorylinositol is acylated to form phosphatidylinositol. Presented in part at the AOCS Spring Meeting, Dallas, April, 1975.     

Selectivity in incorporation,utilization and retention of oleic and linoleic acids by human skin fibroblasts

Fetal human fibroblasts were grown in culture medium containing 10% fetal bovine serum supplemented with [1-¹⁴C] linoleate or [1-¹⁴C] oleate. At all concentrations of exogenous fatty acids, the incorporation of oleate was greater than that of linoleate. With increased medium fatty acid concentrations, linoleate in triacylglycerol (TAG) could be increased from 13 to 75% of the total incorporated; at each concentration, relatively more linoleate than oleate was in TAG. When the cells were exposed to exogenous oleate/linoleate mixtures, the composition of the mixture determined the extent of incorporation of both fatty acids. When the mixture was primarily linoleate, scarce oleate was used preferentially for phospholipids (PL); no such specificity for scarce linoleate was observed. Addition of exogenous fatty acids resulted in a shift of previously incorporated¹⁴C fatty acids from phospholipid into TAG; retention of oleate in PL was greater than that of linoleate. Incorporation of oleate into phospholipids was also higher than that of linoleate from exogenous fatty acid mixtures which were 80% saturated. It is suggested that normal human fibroblasts have adapted to the low levels of exogenous polyunsaturated fatty acids in culture media by increased use of oleate in phospholipid. Even when the cells aresupplemented with linoleate, the preferential use of oleate in phospholipid groups is retained. Presented in part at the ASBC Meeting, Dallas, april 1979.     

Oxysterol mediated changes in fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells

We studied the actions of oxysterols on fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells. Cultures were labeled with [1-¹⁴C] arachidonate or [1-¹⁴C]oleate. During a 24-hr incubation, 25-or 22R-hydroxycholesterol enhanced the incorporation of label into triglycerides, concomitant with a reduction in the labeling of phospholipids. Cholestantriol or 20-hydroxycholesterol had the opposite effects. They caused a higher incorporation of radiolabel into phospholipids and a reduction of labeling of triacylglycerols. Similar changes were seen in cells labeled with [1-¹⁴C]acetate. Therefore, we conclude that oxysterols can promote changes in the distribution of fatty acids between neutral lipids and phospholipids through mechanisms that still need to be clarified.     

Triacylglycerol synthesis in the oleaginous yeastCandida curvata D

Low rates of triacylglycerol (TAG) biosynthesis were observed in cell-free extracts ofCandida curvata, but rates were increased up to 10-fold by adding either α- or β-cyclodextrins. Spheroplasts, whole or gently disrupted, had higher rates of incorporation of both [U-¹⁴C]glycerol 3-phosphate or [1-¹⁴C]oleate into triacylglycerol and the intermediates of its biosynthesis: lysophosphatic acid, phosphatidic acid and diacylglycerol. Fatty acyl-CoA synthetase was highest with palmitate, oleate and linoleate but was some 6- to 8-fold lower with stearate. Stearate and stearoyl-CoA were poorly incorporated into lipids. Subcellular fractionation of the spheroplasts into mitochondrial, microsomal, lipid bodies and supernatant fractions diminished the rates of¹⁴C incorporation of oleate into triacylglycerol. By comparing the relative specific activities for each activity in each fraction, the fatty acyl-CoA synthetase activity appeared mainly in the lipid bodies, and that for phosphatidic acid formation was mainly in the mitochondrion; other activities were too weak and too dispersed for accurate assessment of their location. Recombining all the subcellular fractions restored triacylglycerol synthesizing activity. Omitting any single fraction from the mixture did not result in restoration of triacylglycerol synthesizing activity. Starvation of the yeast, which leads to utilization of the endogenous lipid reserves, stimulated fatty acyl-CoA synthetase activity, but diminished phosphatidic acid and triacylglycerol biosynthesis indicating probable induction of β-oxidation in the peroxisomes and repression of lipid biosynthesis.     

Oleate acutely stimulates the secretion of triacylglycerol by cultured rat hepatocytes by accelerating the emptying of the secretory compartment

The acute effects of addition of oleate on the rate of triacylglycerol (TAG) secretion by cultured rat hepatocytes were studied by monitoring the use of endogenous (¹⁴C-prelabeled) acyl moieties and exogenous (³H-labeled) oleate for the synthesis of secreted TAG simultaneously. Inclusion of exogenous oleate in the medium stimulated the secretion of the endogenous ¹⁴C-labeled acyl moieties by 55–100%. To find out whether the stimulation was due to increased endogenous TAG mobilization or an increased rate of processing of TAG within the endoplasmic reticulum (ER) secretory machinery, use was made of the inhibition of apolipoprotein B (apoB) synthesis (but not degradation) by Ca²⁺ mobilization from the ER. Inhibition of apoB synthesis stopped entry of acyl moieties (from endogenous and exogenous sources) into the secretory pathway. However, even when entry of acyl moieties into the secretory pathway was totally inhibited, exogenous oleate was still able to stimulate (twofold) the secretion [¹⁴C]TAG, indicating that oleate stimulates the emptying of prelabeled TAG from the secretory compartment at a point distal to apoB synthesis and nascent particle formation. These data indicate that exogenous oleate, besides providing additional acyl moieties for incorporation into secreted TAG, stimulates the secretion of endogenous TAG in a manner (i) that is independent of effects on apoB synthesis and/or degradation and (ii) that involves the enhanced processing of TAG resident within the ER secretory pathway.     

Effect of bovine serum albumin on monoacyl- and diacylglycerol 3-phosphate formation in mitochondrial and microsomal fractions of rabbit hearts

The formation of monoacyl- and diacylglycerol 3-phosphate (P) by rabbit heart mitochondrial and microsomal fractions was studied by varying the concentration of acyl-CoA and that of bovine serum albumin in the assay system. The two subcellular fractions were prepared by the conventional differential centrifugation technique. The optimal concentration of acyl-CoA for both mitochondrial and microsomal acylation of glycerol 3-P was shifted to a higher range of acyl-CoA concentrations by greater amounts of albumin. A similar shift in the acyl-CoA concentration-enzyme activity relationship was observed in the acylation reaction of 1-palmitoylglycerol 3-P by the heart microsomes. The addition of albumin increased slightly the rate of diacylglycerol 3-P accumulation but increased greatly the rate of monoacylglycerol 3-P accumulation at any concentration of acyl-CoA; the effect was observed with mitochondrial or microsomal fraction as the crude enzyme source. Moreover, palmitoyl-CoA and linoleoyl-CoA served equally well as the acyl donor for the acylation reaction. However, relatively more monoacyl- than diacylglycerol 3-P was accumulated in the assays with rabbit heart mitochondrial fraction in the presence of albumin, whereas more diacylthan monoacyglycerol 3-P was formed by the microsomal fraction. As a result, the microsomal diacyl: monoacyl-glycerol 3-P ratio was invariably greater than the mitochondrial ratio at a given concentration of acyl-CoA and albumin. This work was supported by grants from the Medical Research Council of Canada and Ontario Heart Foundation.