Regulation of 12-hydroxyeicosatetraenoic acid synthesis by acetyl-LDL in mouse peritoneal macrophages

The mechanism for the regulation of 12-hydroxyeicosatetraenoic acid (12-HETE) production by cholesterol-rich macrophages was investigated. beta-VLDL and acetyl-LDL, lipoproteins which result in cholesterol accumulation in macrophages, stimulated 12-HETE secretion. Lipoproteins which do not induce cholesterol accumulation, such as low- and high-density lipoproteins, did not. Cell-free homogenates from cholesterol-rich macrophages had significantly more 12-lipoxygenase activity than homogenates from unmodified cells. Preincubating homogenates prepared from unmodified macrophages with acetyl-LDL, LDL or multilamellar liposomes containing total lipids from acetyl-LDL but not apoproteins significantly increased 12-lipoxygenase activity. This stimulatory effect was caused by the phospholipid moiety of the lipoprotein. 12-HETE synthesis was not increased in macrophages enriched 6-fold in unesterified cholesterol. Acetyl-LDL stimulated 12-HETE synthesis in macrophages in which cholesteryl ester accumulation was prevented by inhibiting acylcoenzyme A:cholesterol acyltransferase activity. When binding of acetyl-LDL to its receptor was decreased by increasing concentrations of dextran sulfate, or when lysosomal metabolism of the lipoprotein was prevented by chloroquine, 12-HETE production significantly decreased. Moreover, the combination of inhibiting acetyl-LDL binding and degradation completely blocked the stimulation of 12-HETE synthesis by acetyl-LDL. The data indicate that acetyl-LDL must enter the macrophage and be partially degraded to regulate 12-HETE synthesis. The regulation is independent of cholesterol accumulation but is related to the entering lipoprotein phospholipid.     

Effect of cholesterol deficiency on the composition of phospholipids and fatty acids of the housefly, Musca domestica

The housefly larvae were grown in the aseptic diet containing 0.56 μmole cholesterol/g wet weight of diet (control) and 0.05 μmole cholesterol/g wet weight of diet (deficient). The effects of cholesterol deficiency upon the phospholipid composition and fatty acids of the total phospholipid and triglyceride fractions from the lipid extract of the various larval tissues, whole larva, and in both sexes of adults 4 days after eclosion were examined. The total sterol and phospholipid contents (expressed relative to the wet weight of the insect) of the control and deficient insects at the larval and adult stages were analysed and molar ratios compared. The results suggest that cholesterol deficiency reduced the free sterol content of the larvae and adult insects to approximately 25% of the content of the control insects. However, cholesterol deficiency did not effect the phospholipid content during larval and adult stages when compared to that of control insects. Though the larvae reared on the cholesterol deficient diets did not show a profound alteration in the phospholipid composition, a marked increase in the ratio of phosphatidylcholine to phosphatidylethanolamine of the larval fat body and composite gut fraction were noticed. The cholesterol deficiency induced significant changes in the fatty acid composition of the phospholipid fraction of the insect. The ratio of unsaturated fatty acids to saturated fatty acids of the phospholipid fractions decreased significantly due to cholesterol deficiency in the whole larvae and in both sexes of adult flies. The data indicates that cholesterol deficient insects compensated for the lack of cholesterol by increasing saturated fatty acids preferentially in the phospholipid fraction of the lipids for the maintenance of proper membrane fluidity.     

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.     

Protein deficiency and age related alterations in rat peritoneal macrophage lipids

The effects of dietary protein restriction and age on the thioglycollate elicited peritoneal macrophage lipid constituents were studied. Impact of subtle changes in lipid components on macrophage functions have been assessed. Lipid profiles of macrophages recovered from rats fed 20 and 4% protein diets and stock diet fed rats (0 and 3 wk) were comparable qualitatively. Quantitative analysis however revealed significant decrease in phospholipids (30–40%) and consequent elevation of cholesterol/phospholipid molar ratios in the protein depleted and young rats (0 wk), compared to the protein fed groups. The protein deficient and the young rats also exhibited accumulation of certain neutral lipids and reduction in triglycerides. Analysis of fatty acid methyl esters of macrophage phospholipids revealed the predominance of long chain polyunsaturated fatty acids even when oleic (C_18:1) and linoleic (C_18:2) formed the bulk of unsaturated fatty acids in the diet. However, the long chain poly unsaturated fatty acid content, particularly the docosahexaenoic acid (C_22:6n-3) was greatly reduced in the protein depleted and 0 wk rats. Observed changes in the long chain polyunsaturated fatty acids of macrophage phospholipids may be of physiological significance as they modulate the immunological functions of the cell.     

Metabolism of low-density lipoprotein-proteoglycan complex by macrophages: further evidence for a receptor pathway

Earlier, we (Vijayagopal, P., et al. (1985) Biochim. Biophys. Acta 837-251) have shown that complexes of plasma low-density lipoproteins (LDL) and arterial chondroitin sulfate-dermatan sulfate proteoglycan aggregate promote LDL degradation and cholesteryl ester accumulation in mouse peritoneal macrophages. Further studies were conducted to determine whether LDL-proteoglycan complex is metabolized by a receptor-mediated process. Native proteoglycan aggregate was isolated from bovine aorta by associative CsCl isopycnic centrifugation. Complex of 125I-labeled LDL and proteoglycan aggregate formed in the presence of 30 mM Ca2+ was incubated with macrophages, and the binding at 4 degrees C and degradation at 37 degrees C of 125I-labeled LDL in the complex was monitored. Both binding and degradation of the complex were specific and saturable, suggesting that the processes are receptor mediated. The Kd for binding was 23 micrograms LDL protein per ml in the complex. Degradation of 125I-labeled LDL-proteoglycan complex was not suppressed by preincubation of macrophages with excess unlabeled complex, suggesting that the receptor for the complex is not subject to down regulation. Both binding and degradation of the complex and the resultant stimulation of cholesteryl ester synthesis were inhibited by limited treatment of cells with low doses of trypsin and pronase, indicating that the binding sites are protein or glycoprotein in nature. Binding was not inhibited by an excess of native LDL and beta-VLDL and exhibited only partial competition by excess unlabeled acetyl-LDL; however, polyinosinic acid, fucoidin and dextran sulfate, known inhibitors of acetyl-LDL binding and degradation in macrophages, did not affect LDL-proteoglycan complex binding and degradation. Similarly, excess unlabeled LDL-proteoglycan complex produced only partial inhibition of the binding and degradation of 125I-labeled acetyl-LDL by macrophages, suggesting that the binding sites for acetyl-LDL and LDL-proteoglycan complex are probably not identical. These studies provide evidence for a receptor-mediated pathway for the metabolism of LDL-proteoglycan complex in macrophages.     

Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages.

Cholesteryl ester-loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions. Low density lipoprotein (LDL) receptor-mediated endocytosis of native LDL is a relatively poor inducer of macrophage cholesteryl ester accumulation. However, the data herein show that in the presence of a very small amount of sphingomyelinase, LDL receptor-mediated endocytosis of 125I-LDL was enhanced and led to a 2-6-fold increase in 125I-LDL degradation and up to a 10-fold increase in cholesteryl ester accumulation in macrophages. The enhanced lipoprotein uptake and cholesterol esterification was seen after only approximately 12% hydrolysis of LDL phospholipids, was specific for sphingomyelin hydrolysis, and appeared to be related to the formation of fused or aggregated spherical particles up to 100 nm in diameter. Sphingomyelinase-treated LDL was bound by the macrophage LDL receptor. However, when unlabeled acetyl-LDL, a scavenger receptor ligand, was present during or after sphingomyelinase treatment of 125I-LDL, 125I-LDL binding and degradation were enhanced further through the formation of LDL-acetyl-LDL mixed aggregates. Experiments with cytochalasin D suggested that endocytosis, not phagocytosis, was involved in internalization of sphingomyelinase-treated LDL. Nonetheless, the sphingomyelinase effect on LDL uptake was macrophage-specific. These data illustrate that LDL receptor-mediated endocytosis of fused LDL particles can lead to foam cell formation in cultured macrophages. Furthermore, since both LDL and sphingomyelinase are present in atherosclerotic lesions and since some lesion LDL probably is fused or aggregated, there is a possibility that sphingomyelinase-treated LDL is a physiologically important atherogenic lipoprotein.     

Lipid composition of the pea aphid,Acyrthosiphon pisum (Harris) (homoptera: Aphididae), reared on host plant and on artificial media

Polar and neutral lipids and their constitutive fatty acids were quantified in the pea aphid, Acyrthosiphon pisum (Harris), grown on host plant or on a lipid free artificial diet. The results were compared to determine if lipids were involved in the suitability of the diet for continuous rearing of this A. pisum biotype. For apterous adults grown on plants, the lipids were characterized by a low amount of neutral lipids (2.5% weight/fresh weight) almost entirely (96.4%) composed of hexanoyl and sorboyl dimyristin. These storage lipids were higher in the alatae (3.8%), probably correlated with potential flight activity. The phospholipid amounts were identical in these two morphs (1.3–1.4% weight/fresh weight), comprised mainly of phosphatidylethanolamines (52%) and phosphatidylcholines (40.6%). These phospholipids contained a still unidentified fatty acid, with a retention time close to that of linolenic acid and synthesized by the aphid or its bacterial symbionts (not found in plants). The apterous adult aphids reared on an artificial diet showed an accumulation of neutral lipids (8.9% for the first generation); this increase was shown to be slightly greater for the hexanoyl and sorboyl triglycerides. In contrast, the phospholipids decreased in aphids reared on an artificial diet (1.1% and 0.9%, respectively, for first and second generation), correlated with a phospholipid fatty acid profile significantly deficient in C18:3 and in the unidentified peculiar fatty acid. These phospholipids are essential components of biological membranes and a diet-driven deficient synthesis in some of their components may result in the observed symptoms. © 1992 Wiley-Liss, Inc.     

Inhibition of mouse macrophage degradation of acetyl-low density lipoprotein by interferon-gamma

In vitro, metabolism of modified forms of low density lipoprotein (LDL) by macrophages via the acetyl-LDL receptor pathway promotes the massive cellular accumulation of lipid. It has been postulated that in vivo this contributes to foam cell formation in the atherosclerotic lesion. Recent studies have shown that arterial wall cells in vitro can secrete a number of cytokines, several of which have been reported to modulate macrophage cell function. Thus, cytokines have the potential to modulate the acetyl-LDL receptor pathway and to influence the rate of foam cell generation. To study the regulation of this pathway by cytokines, the effect of cytokines on the degradation of acetyl-LDL protein by mouse peritoneal macrophages was examined. Initially, supernatant from stimulated lymphocytes was used as a source of cytokines. Macrophages preincubated with supernatants obtained after the stimulation of T-cell helper type 1 (Th1) clone HDK-1 or BALB/c spleen cells degraded acetyl-LDL at a slower rate, whereas supernatant from stimulated T-cell helper type 2 (Th2) clone D-10 had no effect. Comparison of the lymphokine profiles showed that spleen and HDK-1 cells secreted several lymphokines in common including significant levels of interferon-gamma. Interferon-gamma was then directly shown to be inhibitory; an anti-interferon-gamma monoclonal antibody blocked the HDK-1-mediated inhibition by 70% and the addition of recombinant interferon-gamma (IFN-gamma) to macrophages inhibited the specific degradation of acetyl-LDL in a dose- and time-dependent manner with a maximum suppression to approximately 40% of control. The inhibition was not accompanied by an increase in the amount of cell-associated acetyl-LDL and was not due to cell death nor could it be accounted for by the presence of endotoxin. To study the mechanism of the inhibition, the effects of IFN-gamma on the itinerary of acetyl-LDL and its receptor were examined. IFN-gamma decreased specific acetyl-LDL binding only to a small degree, and the rate of lysosome-mediated degradation was not affected. The principal alteration was in the rate of transport to the lysosome which was markedly slowed. Since the receptors eventually returned to the surface to maintain a steady state, and there was not an increase in cell-associated lipoprotein, there must be other changes in the itinerary that were not identified with the techniques used. Thus, the receptor cycle is being regulated at a discrete point. IFN-gamma also suppressed the LDL receptor pathway in macrophages, but this pathway was not affected by IFN-gamma in mouse fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of Free Fatty Acid Effects on Hepatocyte Insulin Receptor Binding and Processing

We determined whether the paimitate effects on hepatocyte insulin receptor binding and post-receptor trafficking were mediated by accelerated mitochondrial (β-oxidation or accumulation of intracellular fatty acyl-CoA derivatives and possibly protein acyiation. Preincubation of hepatocytes with moderate concentrations of paimitate (0.5 mM) resulted in a 23% decline in cell-surface binding and proportional decreases in receptor-mediated insulin internalization and degradation. Brief pretreatment of hepatocytes with the carnitine palmityltransferase-I inhibitor, methyl paimoxirate (MP), prevented 70% of the paimitate effects. At higher paimitate concentrations (2.0 mM), cell-surface binding was reduced by 34%, whereas internalization of the receptor complex was reduced by 78%. These effects were only partially prevented by MP pretreatment. Receptor-mediated insulin degradation increased by 34% and was uninfluenced by MP pretreatment. Octanoate, which is rapidly shunted into mitochondrial oxidation, produced a dose-dependent reduction in insulin binding, with proportional decreases in internalization and degradation. Similarly preincubation with 2.0 mM oleate, which, unlike palmitate, is not known to produce protein acylation, resulted in proportional decreases in insulin receptor binding and receptor-mediated internalization and degradation. High concentrations of octanoate or oleate (2.0 mM) did not reproduce the additive postreceptor effects of palmitate. We conclude that the receptor and post-receptor effects of moderate palmitate concentrations are closely linked to accelerated fatty acid oxidation. The post-receptor effects observed at higher concentrations involve other mechanisms, possibly relating to intracellular levels of palmityl-CoA derivatives.     

A low density lipoprotein-sized particle isolated from human atherosclerotic lesions is internalized by macrophages via a non-scavenger-receptor mechanism

A lipoprotein particle designated A-LDL, which contains apolipoprotein B (apoB) and which is the size of plasma low density lipoproteins (LDL), was isolated from homogenates of human aortic athersclerotic plaques by a combination of affinity chromatography and gel-filtration. Compared to plasma LDL, A-LDL was more electronegative, its hydrated density was lower and more heterogeneous, and its protein-to-lipid ratio was lower. In addition, apoB in A-LDL was highly degraded, and A-LDL was recognized by mouse peritoneal macrophages (MPM) as indicated by its ability to stimulate cholesterol esterification. Cholesterol esterification was saturable with an apparent Km of 100 micrograms of A-LDL cholesterol/ml. Stimulation of cholesterol esterification was linear with time, leading to extensive accumulation of cholesteryl ester in MPM over a 48-hr time interval. The uptake or degradation of acetyl-LDL (radiolabeled either in the protein with 125I or hydrophobic core with [3H]cholesteryl ether) was markedly decreased by excess unlabeled acetyl-LDL but not by A-LDL, and excess acetyl-LDL did not inhibit the uptake or degradation of labeled A-LDL. However, a 10-fold excess of A-LDL also failed to inhibit the uptake of labeled A-LDL. This finding was consistent with the observation that, unlike the saturable stimulation of cholesterol esterification in MPM induced by A-LDL, the uptake of cholesteryl ether-labeled A-LDL was almost linear over a 0-400 micrograms cholesterol/ml range. This discrepancy between dose response curves for A-LDL, which did not occur for acetyl-LDL, could be eliminated by a 24-hr postincubation period in the absence of lipoprotein, suggesting that A-LDL is catabolized less efficiently than acetyl-LDL following internalization. In summary, we conclude that A-LDL uptake by MPM occurs via a low affinity-high capacity process. Although the uptake of A-LDL is not readily saturated, it is of sufficient affinity to lead to lipid loading of macrophages even when A-LDL is present at relatively low concentrations. If these mechanisms are operative in vivo, they could explain how foam cells in human fatty streak lesions develop.     

Transforming growth factor-beta 1 inhibits scavenger receptor activity in THP-1 human macrophages.

The macrophage scavenger receptor, a 220-kDa trimeric membrane glycoprotein, mediates the internalization of modified forms of low density lipoprotein (LDL) such as acetyl-LDL and oxidized-LDL and thus is likely to play a key role in atheroma macrophage foam cell formation. In addition, recent evidence suggests that the scavenger receptor may be an important macrophage binding site for lipopolysaccharide involved in lipopolysaccharide scavenging by macrophages. However, little is known about the regulation of this important receptor. We now report that the induction of scavenger receptor activity (as measured by acetyl-LDL stimulation of intracellular cholesterol esterification) seen in phorbol ester-differentiated THP-1 human macrophages was completely suppressed to the level seen in undifferentiated THP-1 monocytes by picomolar concentrations of transforming growth factor-beta 1 (TGF-beta 1). 125I-Acetyl-LDL degradation was inhibited in a dose-dependent manner by TGF-beta 1, with maximal inhibition (approximately 70%) occurring at 24 pM TGF-beta 1. Scatchard analysis revealed that TGF-beta 1 treatment resulted in a approximately 2-fold decrease in receptor number, and Northern blot analysis of RNA isolated from differentiated THP-1 macrophages demonstrated approximately 2-fold less scavenger receptor mRNA in TGF-beta 1-treated cells compared with that in macrophages not treated with TGF-beta 1. Since TGF-beta 1 is thought to be present in both atherosclerotic and inflammatory lesions, the above findings may have physiological relevance regarding the regulation of atheroma foam cell formation and/or the regulation of lipopolysaccharide clearance by macrophages.     

Phospholipid supplementation reverses behavioral and biochemical alterations induced by n-3 polyunsaturated fatty acid deficiency in mice

This study investigated the effects of a diet deficient in alpha-linolenic acid followed or not by supplementation with phospholipids rich in n-3 polyunsaturated fatty acids (PUFA) on behavior and phospholipid fatty acid composition in selected brain regions. Three weeks before mating, two groups of mice were fed a semisynthetic diet containing both linoleic and alpha-linolenic acid or a diet deficient in alpha-linolenic acid. Pups were fed the same diet as their dams. At the age of 7 weeks, a part of the deficient group was supplemented with n-3 PUFA from either egg yolk or pig brain phospholipids for 2 months. In the open field, rearing activity was significantly reduced in the deficient group. In the elevated plus maze (anxiety protocol), the time spent on open arms was significantly smaller in deficient mice than in controls. Using the learning protocol with the same task, the alpha-linolenic acid deficiency induced a learning deficit. Rearing activity and learning deficits were completely restored by supplementation with egg yolk or cerebral phospholipids, though the level of anxiety remained significantly higher than that of controls. There were no differences among the 4 diet groups for either the Morris water maze or passive avoidance. In control mice, the level of 22:6 n-3 was significantly higher in the frontal cortex compared to all other regions analysed. The frontal cortex and the striatum were the most markedly affected by the deficiency. Supplementation with phospholipids restored normal fatty acid composition in brain regions except for frontal cortex. Egg yolk or cerebral phospholipids are an effective source of n-3 PUFA for reversing behavioral changes and altered fatty acid composition induced by a diet deficient in n-3 PUFA.     

Acetyl-LDL stimulates macrophage-dependent plasminogen activation and degradation of extracellular matrix

The ability of acetyl-LDL to stimulate macrophage-dependent plasminogen activation and degradation of extracellular matrix was examined. We have found that expression of plasminogen activator activity in response to the scavenger receptor ligand varied among cell populations. Exposure to acetyl-LDL stimulated plasminogen activator expression by cells which constitutively released low levels of activator. These include a virally transformed macrophage-like cell line (RAW246.7), concanavalin A and C. parvum-activated macrophages. The stimulation of plasminogen activator activity was independent of cellular lipid accumulation since nonlipoprotein inhibitors of acetyl-LDL binding to the scavenger receptor stimulated activator expression in great excess to that observed with acetyl-LDL. In contrast, acetyl-LDL was unable to induce soluble plasminogen activator activity in cells which normally do not express it. These include a macrophage-like cell line (J774A.1) and resident peritoneal macrophages. Furthermore, acetyl-LDL was unable to modulate the copious secretion of activator by inflammatory macrophages elicited with thioglycolate. When macrophages were tested for their ability to degrade smooth muscle cell derived matrix, solubilization by resident, elicited, and activated cells was variously increased in the presence of plasminogen. Furthermore, exposure to acetyl-LDL enhanced plasmin-dependent degradation by resident cells and activated cells, whereas matrix degradation by elicited cells was unaffected.     

Dietary n-6 PUFA deprivation for 15 weeks reduces arachidonic acid concentrations while increasing n-3 PUFA concentrations in organs of post-weaning male rats

Few studies have examined effects of feeding animals a diet deficient in n-6 polyunsaturated fatty acids (PUFAs) but with an adequate amount of n-3 PUFAs. To do this, we fed post-weaning male rats a control n-6 and n-3 PUFA adequate diet and an n-6 deficient diet for 15 weeks, and measured stable lipid and fatty acid concentrations in different organs. The deficient diet contained nutritionally essential linoleic acid (LA,18:2n-6) as 2.3% of total fatty acids (10% of the recommended minimum LA requirement for rodents) but no arachidonic acid (AA, 20:4n-6), and an adequate amount (4.8% of total fatty acids) of α-linolenic acid (18:3n-3). The deficient compared with adequate diet did not significantly affect body weight, but decreased testis weight by 10%. AA concentration was decreased significantly in serum (− 86%), brain (− 27%), liver (− 68%), heart (− 39%), testis (− 25%), and epididymal adipose tissue (− 77%). Eicosapentaenoic (20:5n-3) and docosahexaenoic acid (22:6n-3) concentrations were increased in all but adipose tissue, and the total monounsaturated fatty acid concentration was increased in all organs. The concentration of 20:3n-9, a marker of LA deficiency, was increased by the deficient diet, and serum concentrations of triacylglycerol, total cholesterol and total phospholipid were reduced. In summary, 15 weeks of dietary n-6 PUFA deficiency with n-3 PUFA adequacy significantly reduced n-6 PUFA concentrations in different organs of male rats, while increasing n-3 PUFA and monounsaturated fatty acid concentrations. This rat model could be used to study metabolic, functional and behavioral effects of dietary n-6 PUFA deficiency.     

The processing of ligands by the class A scavenger receptor is dependent on signal information located in the cytoplasmic domain

The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser(21) increased acetyl-LDL metabolism, whereas inactivation of the site at Ser(49) reduced acetyl-LDL uptake. The increase in uptake by the Ser(21) mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser(49) did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, beta-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp(25) inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand.     

Trans fatty acids in hydrogenated fat inhibited the synthesis of the polyunsaturated fatty acids in the phospholipid of arterial cells

Our hypothesis that the trans fatty acids in hydrogenated fat inhibited the synthesis of polyunsaturated fatty acids in the phospholipid of arterial cells was tested with five groups each with six pregnant porcine fed from d 35 of gestation and during lactation. The basal diet contained 2% corn oil (control). The other four diets included the control + 10% butter or 10% hydrogenated fat plus two levels of Mg. Plasma, milk and aortic phospholipid fatty acids, phospholipid composition and calcium content of the aorta from the piglets were determined. At 48 +/- 2 d of age, the aorta phospholipid of piglets from porcine fed hydrogenated fat contained a significantly higher concentration of linoleic acid, less arachidonic acid, and less long chain polyunsaturated fatty acid (PUFA) than did piglets from porcine fed either butterfat or the control diet. Mg had no effect. These changes in composition in piglets from porcine fed hydrogenated fat indicate that trans fat inhibits the metabolic conversion of linoleic acid to arachidonic acid and to other n-6 PUFA. The aortic calcium content data showed a significant interaction of calcium concentration with age. We concluded: 1) that dietary trans fat perturbed essential fatty acid (EFA) metabolism which led to changes in the phospholipid fatty acid composition in the aorta, the target tissue of atherogenesis, 2) this inhibition of EFA to PUFA by the isomeric fatty acids in hydrogenated fat is a risk factor in the development of coronary heart disease.     

Fatty acid composition, eicosanoid production and permeability in skin tissues of rainbow trout (Oncorhynchus mykiss) fed a control or an essential fatty acid deficient diet

Rainbow trout (Oncorhynchus mykiss) were fed either a control diet containing fish oil or an essential fatty acid (EFA) deficient diet containing only hydrogenated coconut oil and palmitic acid as lipid source (93.4% saturated fatty acids) for 14 weeks and the fatty acid compositions of individual phospholipid classes from skin and opercular membrane (OM) determined. The permeability of skin and OM to water and the production of eicosanoids in skin and gills challenged with the Ca²⁺ ionophore A23187 were also measured. Phospholipid (PL) fatty acid compositions were substantially modified in EFA-deficient fish, with increased saturated fatty acids and decreased polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) and eicosapentaenoic acid (EPA), while docosahexaenoic acid (DHA) was largely retained. The onset of EFA deficiency was shown by the appearance of n-9 PUFA, particularly 20:3n-9. The main effects of EFA deficiency on phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were to increase saturated fatty acids and monoenes, especially 16:1 and 18:1, and to decrease EPA and DHA. The content of DHA in phosphatidylserine (PS) was high in control animals (40% in skin and 35% in opercular membrane) and was mostly retained in EFA deficient animals. Arachidonic acid (AA) was the most abundant PUFA esterified to phosphatidylinositol (PI) and was significantly reduced in EFA deficient animals (from 31% to 13% in skin), where a large amount of 20:3n-9 (9% in skin) was also present. Influxes and effluxes of water through skin and opercular membrane were measured in vitro. No differences were detected between rainbow trout fed the control or the EFA deficient diet. 12-Hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHE) could not be detected in skin from control or EFA deficient fish. There was no difference between control and EFA deficient trout in the levels of leukotriene C₄ (LTC₄) and leukotriene C₅ (LTC₅) in skin cells challenged with the calcium ionophore A23187, and of prostaglandin F_2α (PGF_2α), 12-HETE and 12-HEPE in gill cells challenged similarly. Prostaglandin F_3α (PGF_3α) production by ionophore stimulated gill cells was significantly reduced in fish fed the EFA-deficient diet. 14-HDHE produced by gill cells was 3.3 fold higher in EFA deficient fish compared to controls.     

Lipid Composition of the Brain in the Vitamin B12-Deficient Fruit Bat (Rousettus aegyptiacus) with Neurological Impairment

Abstract: The Egyptian fruit bat Rousettus aegyptiacus develops severe vitamin B₁₂ deficiency when fed a diet of fresh peeled fruit and water. In a group of bats fed this diet, B₁₂ concentrations in the serum and brain were low, and neurological impairment, evidenced by deficient or absent hindlimb groping or grasping ability and climbing difficulties, was manifest. Control bats fed the identical diet supplemented with B₁₂ showed no such changes. Fatty acid analysis of whole brain homogenates showed a higher level of 20:4 in the deficient group. Phosphatidylcholine showed a marginally higher percentage of 18:3. The total percentage of branched chain fatty acids of phos-phatidylethanolamine was four times higher in deficient brains, comprising 2% of the total. Sphingomyelin showed a slightly higher percentage of 15:0, and a significantly lower percentage of long chain fatty acids C-24 and longer ( p < 0.01). The compositions of nonhydroxy fatty acids in cerebroside were unchanged. Examination of phospholipids showed that 8.9 ± 0.4% of total phosphorus was present as sphingomyelin in deficient bats, compared with 11.9 ± 1.2% in control animals (p < 0.05). There were no statistically significant differences in the concentrations of total brain lipid, protein, phospholipid, glycosphingolipid, cholesterol and plasmalogen between B₁₂-deficient and control bats.     

Phospholipid hydroperoxide glutathione peroxidase activity and vitamin E level in the liver microsomal membrane: effects of age and dietary alpha-linolenic acid deficiency

Age and diet-induced variations of phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity and alpha-tocopherol concentration in the liver microsomal membrane were studied in male Wistar rats fed a semipurified diet either balanced in n-6 and n-3 polyunsaturated fatty acids (PUFA) (Control) or deprived of alpha-linolenic acid, i.e. n-3 PUFA (Deficient) over two generations. The animals were studied at the age of 6 months (adult) or 24 months (old). Both PHGPx activity and vitamin E level were significantly higher in 24-month old rats as compared to 6-month old rats. By contrast, the thiobarbituric acid reactive substances (TBARS) following stimulated in vitro peroxidation of membrane lipids were markedly lower (P < 0.01) with aging. The fatty acid composition of microsomal membrane phospholipids (PL) was also considerably modified by age. In particular, the levels of arachidonic acid and total n-6 PUFA were lower (P < 0.001) whereas n-3 PUFA levels were higher (P < 0.001) in most PL main classes. The alpha-linolenic acid deficiency markedly influenced these age-related changes. The higher PHGPx activity in the old rats as compared to the adult rats was only significant in those fed the control diet. In the 6-month old rats (but not in the 24-month old rats), the deficient diet led to a higher membrane vitamin E level and to lower TBARS production than the control diet. The results suggest that the nature of dietary PUFA may influence the age-related variations in this pair of membrane antioxidants and also in the fatty acid composition of microsomes.     

Characterization of binding sites for acetylated low density lipoprotein in the rat liver in vivo and in vitro.

Acetylated low density lipoprotein (acetyl-LDL) binding to hepatic membrane proteins of rats was analysed in vitro by ligand blotting. Specific binding could be demonstrated to two hepatic proteins with an apparent mol. wt. of 250 kd and 220 kd. Polyanionic competitors and maleylated bovine serum albumin inhibited the binding of acetyl-LDL effectively. To determine the sites of the catabolism of acetyl-LDL, [131I]-acetyl-LDL was injected intravenously into control rats and rats pre-treated with the known competitors of the acetyl-LDL binding. Distribution of the radiolabelled acetyl-LDL was followed by a scintillation camera. Six minutes after injection, the radioactivity was concentrated in the liver. The competitors and unlabelled acetyl-LDL but not native LDL reduced the hepatic uptake of [131I]acetyl-LDL dramatically. Thus, the sensitivity of the 220- and 250-kd membrane binding sites to the competitors for the acetyl-LDL binding resembled that of the hepatic compartment in vivo. Finally, an application of scintigraphy with radiolabelled low density lipoproteins for diagnostic evaluation of tumor compartments is presented.