Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues.

A triacylglycerol and cholesteryl ester transfer activity has been isolated from rat liver. After homogenization, the liver cells were subfractionated into the 10 000 X g pellet, microsomal fraction and postmicrosomal supernatant. Most of the transfer activity appeared to be associated with the microsomal fraction. Rough and smooth microsomes contained nearly equal transfer activities. When isolated microsomes were subject to proteolytic attack, the transfer activity was not inactivated, unless it had been released from the microsomes prior to proteolytic treatment. This indicates that the activity is probably located within the microsomal vesicles. Similar transfer activities were found in the intestinal mucosa of rats, whereas little or no activity was detected in the brain, heart, kidney, or plasma.

Disappearance and effects of exogenous lipid transfer activity in rats.

These studies were performed to determine the role of plasma lipid transfer activity in the regulation of plasma total and lipoprotein cholesterol in vivo. Partially purified human lipid transfer activity was injected into rats at a level similar to that of normal rabbit plasma, d greater than 1.21. The disappearance of exogenous lipid transfer activity from rat plasma was biphasic, with a 70% loss within 6 h. The remaining 30% was lost with a half-time of about 14 h. In the rat, short-term exposure (6 h) to high levels of lipid transfer activity resulted in a net transfer of cholesteryl esters from high density to d less than 1.019 lipoproteins, without affecting plasma total cholesterol. However, the lipid transfer activity-induced changes in lipoprotein cholesterol were not evident after 24 h, despite the fact that the lipid transfer activity of rat plasma d greater than 1.21 was similar to that of human plasma d greater than 1.21 during the preceding 18 h.

Impaired triacylglycerol catabolism in hypertriglyceridemia of the diabetic, cholesterol-fed rabbit: a possible mechanism for protection from atherosclerosis.

The etiology of the hypertriglyceridemia in alloxan-diabetic rabbits was studied by two independent methods. Production and removal rates of VLDL triacylglycerol were measured in diabetic rabbits by injection of [3H]palmitate-labelled VLDL. Similarly, triacylglycerol total removal rates were determined in non-diabetic rabbits which were infused with Intralipid to mimic the plasma triacylglycerol concentrations of diabetic rabbits. Compared to nondiabetic rabbits, triacylglycerol removal rats were decreased in diabetic rabbits, particularly at higher levels of plasma triacylglycerol. During cholesterol and triacylglycerol supplementation of the diet, post-heparin plasma lipoprotein lipase activity of diabetic rabbits with severe hypertriglyceridemia averaged 36% of that of nondiabetics, suggesting an impaired triacylglycerol removal capacity. Furthermore, plasma triacylglycerol was inversely related to post-heparin plasma lipoprotein lipase activity among diabetic rabbits. VLDL triacylglycerol production increased with increasing plasma triacylglycerol concentration among diabetic cholesterol-fed rabbits with moderately severe hypertriglyceridemia, but reached an apparent plateau among rabbits with plasma triacylglycerol concentrations from approx. 2000-9000 mg/dl. Thus, severe hypertriglyceridemia in this model of insulin deficiency can be attributed only partially to VLDL hypersecretion, whereas a removal defect, resulting in saturation of the triacylglycerol removal mechanism, appears to be largely responsible. The impaired removal of plasma triacylglycerol is also related to the presence of cholesterol predominantly in lipoproteins of increased size. The data support the hypothesis that protection against atherosclerosis in cholesterol-fed diabetic rabbits results from exclusion of very large cholesterol-containing lipoproteins from the arterial wall.

In vivo influx, tissue esterification and hydrolysis of free and esterified plasma cholesterol in the cholesterol-fed rabbit.

The influx of free and esterified cholesterol into various tissues of cholesterol-fed rabbits is calculated from the tissue [3H] cholesterol and [14C] cholesterol content - corrected for radioactivity in contaminating plasma - after a 3--6 h exposure to in vivo-labeled plasma. The plasma free cholesterol was labeled primarily with 3H and the esterified cholesterol with 14C or vice versa. The influx calculation is based on total 3H and 14C in tissues and two linear equations that take into account esterification and hydrolysis of sterol fractions by the tissues. The influx of esterified cholesterol into tissue samples from aorta, heart, small intestine and lung was 10--80 nmol-g--1-h--1, whereas the influx into adrenal, spleen and liver was from 400--2500 nmol--g-1--h-1. The influx of free cholesterol was considerably higher than expected if free and esterified cholesterol had entered the tissues together as part of plasma lipoproteins. This excess of free cholesterol influx can be ascribed to cholesterol exchange between plasma lipoproteins and tissues, which in several tissues amounted to more than 80% of the total free cholesterol influx. From tissue free and esterified cholesterol radioactivity, one can calculate that 20--70% of the newly entered esterified cholesterol was hydrolyzed by various tissues and that most tissues esterified less than 10% of newly entered cholesterol during the experimental period. However, esterification of plasma cholesterol by adrenals averaged 50% of that taken up during a 3-6 h period.

The separation of apolipoprotein D from cholesteryl ester transfer protein.

This study addresses the question of whether cholesteryl ester transfer protein and apolipoprotein D are identical. The data presented show that these two proteins do not co-purify during hydrophobic and cationic exchange chromatography and are readily separated by molecular sieve chromatography or electrophoresis. Furthermore, the precipitation of apolipoprotein D by specific antisera did not diminish the transfer activity of lipoprotein-deficient plasma. We conclude that apolipoprotein D and cholesteryl ester transfer protein have significantly different physicochemical properties.

Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.

A new rat liver phospholipid exchange protein.

The soluble fraction from several mammalian tissue homogenates is known to stimulate phospholipid exchange between cell membrane fractions or artificial vesicles. All phospholipid exchange proteins purified to data exhibit an acidic isoelectric point. Using an assay that measures the transfer of [32P] phosphatidylcholine from liposomes to beef heart mitochondria, we report the presence of a new phospholipid exchange protein with a basic isoelectric point (8.4) in rat liver cytosol. A purification procedure, consisting of pH adjustment to 5.1, gel filtrations on Sephadex G 75 and DE 52 cellulose, isoelectric focusing between a pH of 5 and 10, and gel filtration on Sephadex G-50, yielded a fraction with high phosphatidylcholine exchange activity per mg of protein. This fraction exhibits a major band and two minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the major band (18 700) is close to that for basic exchange protein fraction obtained by gel filtration (17 000). The distribution of basic and acidic exchange proteins differs markedly in various tissues and animal species. About 50 and 35% of phosphatidylcholine exchange activity from rat liver and rat intestine respectively are due to basic phospholipid exchange proteins. In contrast, no basic exchange protein was found in beef heart and only a small amount in beef liver. In the latter organ, less than 10% of phosphatidylcholine exchange activity was due to a basic phospholipid exchange protein fraction.

Net transfer of phospholipid by the nonspecific phospholipid transfer proteins from bovine liver.

The nonspecific phospholipid transfer proteins from bovine liver catalyze net transfer of phosphatidylcholine and phosphatidylinositol from phosphatidylcholine/phosphatidylinositol multilamellar vesicles (9 : 1; mol/mol) to either intact or totally delipidated human high density lipoprotein. Under indentical conditions, the phosphatidylcholine-specific exchange protein from bovine liver and the phosphatidylinositol/phosphatidylcholine exchange protein from beef heart do not enhance the net transfer of labeled phosphatidylcholine or phosphatidylinositol from multilamellar vesicles to delipidated high density lipoprotein, but do catalyze phospholipid exchange between multilamellar vesicles and intact high density lipoprotein consistent with their reported specificities. In the presence of nonspecific transfer proteins net transfer of mass in also observed from phosphatidylcholine unilamellar vesicles to rat liver inner mitochondrial membranes plus matrix. These are the first exchange proteins demonstrated to have net mass transfer capability.

Extensive exchange of rat liver microsomal phospholipids.

Liver microsomal fractions were prepared from rats injected with a single dose of choline [14C]methylchloride or with single or multiple doses of 32Pi. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85--95% exchangeable in 1--2h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer.

Catalytic properties of phospholipid exchange protein from bovine heart.

A protein which catalyzes the exchange of phosphatidylcholine between membranes has been purified from heart tissue homogenates up to 300-fold by acidic pH precipitation, (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography. Binding of the protein to phosphatidylcholine liposomes as measured by Sepharose chromatography was nondetectable. However, isoelectric focusing experiments showed that individual molecules of phosphatidylcholine were transferred from liposomes to the soluble, partially purified protein. Exchange of phospholipid between liposomes and mitochondria was not affected by the presence of moderate amounts of cholesterol in liposomes. A search for competitive inhibitors among moieties similar to phosphatidylcholine failed to show strong binding sites in the hydrophilic part of the substrate. High concentrations of Na+, Ca2+ and Mg2+ impaired the exchange activity.

Comparison of various methods for in vitro cholesteryl ester labeling of lipoproteins from hypercholesterolemic rabbits.

Little or no information is available on biologically valid labeling of hypercholesterolemic plasma lipoproteins with cholesteryl ester. The esterification of labeled unesterified cholesterol in hypercholesterolemic rabbit plasma by the lecithin: cholesterol acyltransferase reaction is inefficient. The use of the d greater than 1.063 plasma fraction for this reaction greatly improves the efficiency, but some labeled unesterified cholesterol remains in the end products. The latter disadvantage can be avoided by the addition to whole plasma of labeled cholesteryl ester dissolved in DMSO or acetone. However, in hypercholesterolemic rabbit plasma only a small fraction of the added cholesteryl ester was associated with lipoproteins. When phosphatidylcholine/cholesteryl ester liposomes were incubated with hypercholesterolemic rabbit plasma for 18-24 h at 37 degrees C the labeled cholesteryl ester was quantitatively incorporated into lipoproteins. Chylomicron-like, cholesteryl ester-rich particles were removed by centrifugation (10(6) g X min) and the subsequently isolated d less than 1.019 and d = 1.019-1.063 (LDL) fractions were injected intravenously into normal and hypercholesterolemic rabbits. The disappearance of d less than 1.019 and LDL cholesteryl ester and the appearance of cholesteryl ester in other lipoprotein fractions was indistinguishable from that of in vivo-labeled lipoproteins. In vivo and in vitro cholesteryl ester-labeled lipoproteins were also compared by measuring the exchangeability of their cholesteryl ester with HDL cholesteryl ester in vitro. Equal exchangeability of the two labels was observed in the d less than 1.019 fraction from which the chylomicron-like particles had been removed. These findings demonstrate that when cholesteryl ester is incorporated by the liposome procedure, the distribution of labeled cholesteryl ester within the lipoprotein complex corresponds closely to that of the in vivo-incorporated labeled cholesteryl ester.

Purification and characterization of human plasma proteins that inhibit lipid transfer activities.

A protein which inhibits cholesteryl ester and triacylglycerol transfer activities was purified from human lipoprotein-deficient plasma by chromatography on phenyl-Sepharose CL-4B, chromatofocusing, Bio-Gel A-0.5m and hydroxylapatite. The inhibitor is a sialoglycoprotein with molecular weight 32 000 and a relatively broad isoelectric region of 3.9-4.3. The inhibitor suppressed triacylglycerol and cholesteryl ester transfer activities to a similar extent. Apolipoprotein A-I, which was separated from the inhibitor by chromatofocusing chromatography, suppressed triacyglycerol transfer more than cholesteryl ester transfer. The percentage reduction of lipid transfer between lipoproteins by the inhibitor was independent of the concentration of transfer protein but was decreased at higher lipoprotein concentrations. The inhibition was not observed during lipid transfer between liposomes. These results indicate that the inhibitor interacts with substrates rather than with the transfer protein.

High de novo synthesis of glycerolipids compared to deacylation-reacylation in rat liver microsomes.

A microsomal system characterized by high flux through the entire de novo pathway from glycerol phosphate to phosphatidylcholine and triacylglycerol has been developed. Optimum synthesis of phosphatidylcholine requires CDPcholine, Mg2+, KCl and a palmitoyl-CoA-generating system containing palmitic acid, ATP and CoA. Incorporation of [14C]glycerol phosphate into phosphatidylcholine/triacylglycerol synthesis ratio decreases as palmitate is increased. Phosphatidylcholine synthesis from glycerol phosphate is stimulated more by palmitate than by other saturated fatty acids; phosphatidylcholine synthesis increases with increasing unsaturation of the added fatty acids. The ratio of incorporation of [3H]palmitate to [14C]glycerol phosphate was determined for phosphatidic acid, diacylglycerol, phosphatidylcholine and triacylglycerol. This ratio is approximately 2 for all diacylglycerolipids and 3 for triacylglycerol. In our system, incorporation of palmitate into microsomal glycerolipid proceeds primarily by the de novo pathway, with minimal fatty acid recycling via deacylation-reacylation.

Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C.

The distribution of phosphatidylinositol and phosphatidylcholine in sonicated phospholipid vesicles (phosphatidylcholine : diphosphatidylglycerol : phosphatidylinositol, 90 : 5 : 5 mol%) has been determined by the use of exchange protein from beef heart and phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Approximately 70% of the phosphatidylinositol in the sonicated vesicles was accessible to the exchange protein and 70--75% was accessible to the phospholipase C. A similar proportion (65%) of the phosphatidylcholine was accessible to the exchange protein suggesting that phosphatidylinositol was not preferentially located in either surface of the phospholipid bilayer. The rate of translocation of both phospholipids was very slow but the rate for phosphatidylcholine (t 1/2 = 4--7 days) appeared to be greater than that for phosphatidylinositol (t 1/2 = 8--60 days). Production of asymmetric vesicles by removing phosphatidylinositol from the outer surface with either exchange protein or phospholipase C did not induce rapid phospholipid translocation.

Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma.

A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin:cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.

Effects of hyperlipidemias in hamsters on lipid transfer protein activity and unidirectional cholesteryl ester transfer in plasma.

Experiments were performed to characterize plasma lipid transfer protein activity (LTA), and the rate of [3H]CE transfer from HDL to lower density lipoproteins in plasma of hamsters. Compared to rabbits, hamster plasma has about one-tenth the level of d greater than 1.21 LTA but a relatively high level of VLDL-triacylglycerols, and a higher fractional rate of HDL-[3H]CE transfer in plasma (in vitro) than predicted by the d greater than 1.21 LTA. Like the rat, hamster plasma contains an inhibitor(s) of LTA; the level of the inhibitor activity in d greater than 1.21 g/ml plasma was similar in normal and hyperlipoproteinemic hamsters. Hypertriglyceridemia in sucrose-fed hamsters did not affect LTA, cholesteryl ester transfer or the plasma level of HDL-CE. However, a comparable degree of hypercholesterolemia was associated with a 122% increase in plasma d greater than 1.21 LTA and a 63% increase in the fractional rate of [3H]CE transfer from HDL to lower density lipoproteins in plasma. Cholesterol feeding in hamsters was associated with increased plasma levels of LDL-cholesterol and, to a lesser extent, with VLDL- and IDL-cholesterol.

Cholesteryl ester exchange protein in human plasma isolation and characterization.

A protein catalyzing the exchange of cholesteryl esters among the lipoproteins was found in human plasma. A rapid method for assaying this activity was developed based on the transfer of radioactive cholesteryl esters from low density lipoprotein with MnCl2 in the presence of phosphate. Fractionation of plasma through a combination of ammonium sulfate precipitation, ultracentrifugation at p = 1.25, and chromatography on Phenyl-Sepharose, CM-cellulose, and concanavalin A-Sepharose, yielded a preparation purified 3500-fold compared to the starting plasma. The exchange protein was found to be a glycoprotein with an isoelectric point of 5 and apparent molecular weight of 80 000. On the basis of these properties and its immunological characteristics the exchange protein was judged to be distinct from any of the known apolipoproteins. This protein could also be separated from plasma phosphatidylcholine cholesterol acyl-transferase on DEAE-cellulose. The exchange protein did not appear to influence cholesterol esterification in lipoproteins by phosphatidylcholine cholesterol acyl-transferase, and the latter had no effect on the transfer of low density lipoprotein cholesteryl esters to high density lipoprotein. The exchange protein did not esterify cholesterol or hydrolyze cholesteryl esters in lipoproteins.

Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes.

The exchange of phosphatidylcholine between (32P)phosphatidylcholine lipososomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of (23P)phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of the initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homofwnwoua pool od amLL single walled vesicles. Treatment of phosphatidyl (14C) choline liposomes with phospholipase D (phosphatidylcholine phosphantidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed cound not by hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of phosphatidylcholine from the inner to the outer layer of the liposome bilayer.

Exchange of retinyl and cholesteryl esters between lipoproteins of rabbit plasma.

Normal or hypercholesterolemic rabbit plasma stimulates the transfer of retinyl ester as well as cholesteryl ester from rabbit lymph chylomicrons, chylomicron remnants or from cholesteryl ester-rich plasma VLDL to the d greater than 1.019 lipoprotein fractions. The presence of p-chloromercuriphenylsulfonate does not inhibit the transfer of these esters. Partially purified lipid transfer protein from rabbit or from human plasma also accelerates the transfer of the above esters. Whereas the rabbit plasma transfer protein preferentially accelerates the transfer of retinyl ester, the human plasma transfer protein appears to have a somewhat greater stimulating effect on the transfer of cholesteryl ester from low- to high-density lipoproteins.

Stimulation by acidic phospholipids of protein-catalyzed phosphatidylcholine transfer.

1. The catalyzed transfer of phosphatidylcholine from unilamellar liposomes to mitochondria by phospholipids exchange protein from beef heart or from beef liver is stimulated by the presence of up to 20 mol% acidic phospholipid (phosphatidylinositol or phosphatidic acid) in the liposome. Co-sedimentation of liposomes with mitochondria increases with increasing mol% acidic phospholipid. 2. The catalyzed transfer of phosphatidylcholine from unilamellar liposomes to multilamellar vesicles by beef heart or beef liver exchange proteins is also stimulated by the presence of acidic phospholipid. No co-sedimentation of negatively charged transfer of phosphatidylcholine from multilamellar vesicles to unilamellar liposomes by phospholipid exchange protein from beef heart or beef liver reaches a maximum at 7.5% phosphatidylinositol in the liposomes. Inhibition of phosphatidylcholine transfer was observed at levels of liposome phosphatiylinositol of greater than 15 mol% only in the presence of beef liver exchange protein. 4. Changes in the surface charge of liposomes by the addition of acidic phospholipid were verified by a novel application of polyvinylchloride block electrophoresis that allows the direct measurement of the relative electrophoretic mobility of sonicated vesicles.