The role of plasma membranes in the transfer of non-esterified cholesterol to the acyl-CoA: Cholesterol acyltransferase substrate pool in liver microsomal fraction

The incubation at 37°C of rat-liver microsomal fraction followed by re-isolation of the treated microsomal vesicles results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The rate of this increase was higher in the microsomal fraction from rats fed cholesterol-supplemented diet or starved overnight as compared with that in the microsomal fraction from rats fed standard diet. The presence of a plasma membrane preparation in the incubation mixture also resulted in a time-dependent increase in acyl-CoA: cholesterol acyltransferase activity at a rate that was dependent on the concentration of plasma membranes. During the incubation of the microsomal fraction in the presence of phosphatidylcholine liposomes, cholesterol is transferred from the microsomal to liposomal vesicles. This transfer followed first-order kinetics with respect to cholesterol concentration in the donor with a rate that increased with the concentration of liposomes in the incubation mixture. The presence of phospholipid was also associated with a decrease in the activity of the acyltransferase that was related to the concentration of phospholipid in the incubation mixture. The incubation of the microsomal fraction in the presence of phosphatidylcholine-cholesterol liposomes resulted in a time-dependent and concentration-dependent transfer of liposomal cholesterol to the microsomal fraction and the acyltransferase substrate pool. The measurement of the rate of transfer of liposomal cholesterol to the microsomal vesicles and to the acyltransferase substrate pool at various temperatures showed that activation energies for the two processes are similar. Similar to these values was also the activation energy for the increase in acyl-CoA: cholesterol acyltransferase activity due to preincubation in the absence of artificial membrane vesicles. The present results suggest that there is, under the present conditions, a time-dependent and temperature-dependent flow of cholesterol from plasma membranes to the acyltransferase substrate pool and that this flow is either diverted in the presence of phospholipid liposomes or increased in the presence of cholesterol-phospholipid liposomes.     

Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles.

Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.     

On the mechanism of the modulation in vitro of acyl-CoA:cholesterol acyltransferase by progesterone.

The assay of acyl-CoA:cholesterol acyltransferase (ACAT) in the presence of progesterone resulted in a lower enzyme activity and this inhibition was dependent on the concentration of steroid in the assay mixture. The incubation at 37 degrees C of rat liver microsomal fraction followed by the re-isolation of treated microsomal vesicles and the assay of ACAT resulted in a pre-incubation-time-dependent increase in the activity of the enzyme. This rate of increase was inhibited by the presence of progesterone in the pre-incubation mixture. The incubation of the microsomal fraction in the presence of cholesterol/phosphatidylcholine liposomes, followed by the re-isolation of the treated microsomal vesicles and assay of ACAT, resulted in time-dependent and liposomal cholesterol-concentration-dependent transfer of cholesterol to microsomal vesicles and in an increase in the activity of ACAT. The presence of progesterone during pre-incubation had no effect on the rate of transfer of liposomal cholesterol to the microsomal vesicles. However, progesterone decreased the rate of change in ACAT activity. This effect can be attributed to progesterone associated with treated microsomal vesicles and present during the enzyme assay. Consistent with this, the presence of progesterone has no effect on the size of the non-esterified cholesterol pool that acts as substrate for ACAT. The size of the ACAT substrate pool was modulated in vitro or in vivo and ACAT activity was assayed in the presence of various concentrations of progesterone. The data suggest that the interaction of the steroid with ACAT is at a site other than the catalytic site and that changes in the size of the substrate pool are associated with an increase in ACAT activity, but do not result in changes in the conformation of the enzyme or in co-operative transitions of the enzyme.     

Acyl-coenzyme A: cholesterol acyltransferase. Transfer of cholesterol to its substrate pool and modulation of activity

The preincubation at 37 degrees C of rat liver microsomal fraction, followed by re-isolation of the treated vesicles, results in a time-dependent increase in the activity of acyl-CoA: cholesterol acyltransferase. The presence of cholesterol-phospholipid (1:1, mol/mol) liposomes results in higher rate of increase in activity and under these conditions the rate of increase is liposomal cholesterol concentration-dependent. The preincubation of the microsomal fraction in the presence of [3H]cholesterol-phospholipid liposomes results in transfer of [3H]cholesterol to the re-isolated microsomal vesicles and this transfer follows first-order kinetics in respect to the donor concentration. These preincubations result also in a time-dependent and liposomal cholesterol concentration-dependent increase in the incorporation of [3H]cholesterol into the cholesteryl oleate produced on assay of cholesterol acyltransferase activity. From specific radioactivity data of the cholesteryl esters synthesised on assay of cholesterol acyltransferase in treated microsomal preparations, the rate of liposomal [3H]cholesterol equilibration with the cholesterol acyltransferase substrate pool can be calculated. The half-time of this transfer decreased with the concentration of liposomal cholesterol present during the preincubation. The activation energy for the transfer of liposomal cholesterol to the cholesterol acyltransferase substrate pool was 87.9 kJ/mol and was independent of the concentration of liposomal cholesterol. The activation energy for the rate of increase of total cholesteryl oleate was similar to this value for low concentrations of liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The data suggest that under the present conditions, the time-dependent and temperature-dependent increase in cholesterol acyltransferase activity is due to the transfer of non-esterified cholesterol from other microsomal and/or liposomal vesicles to the vesicles that contain the enzyme and therefore to increased availability of substrate.     

Annexin I-mediated vesicular aggregation: mechanism and role in human neutrophils.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.     

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.     

Studies on acyl-coenzyme A: cholesterol acyltransferase activity in human liver microsomes

The aim of the present study was to characterize the acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity in human liver microsomes. Liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In 34 patients the enzyme activity of the microsomal fraction averaged 6.6 +/- 0.7 (mean +/- SEM) pmol.min-1.mg protein-1 in the absence of exogenous cholesterol. Freezing of the liver biopsy in liquid nitrogen increased the enzyme activity five- to sixfold. Similarly, freezing of the microsomal fraction prepared from unfrozen liver tissue increased the enzyme activity about twofold. These results may help to explain previous disparate results reported in the literature. The enhanced ACAT activity obtained by freezing was at least partly explained by a transfer of unesterified cholesterol to the microsomal fraction and possibly also by making the substrate(s) more available to the enzyme. Preincubation of the microsomal fraction, prepared from unfrozen liver tissue, with unlabeled cholesterol increased the enzyme activity about fivefold. This finding indicates that hepatic ACAT in humans can also utilize exogenous cholesterol as substrate. Addition of cholesterol to frozen microsomes prepared from unfrozen liver tissue increased the ACAT activity two- to threefold, whereas addition of cholesterol to microsomes prepared from frozen liver tissue did not further increase the enzyme activity. No evidence supporting the concept that ACAT is activated-inactivated by phosphorylation-dephosphorylation could be obtained by assaying the enzyme under conditions similar to those during which the human HMG-CoA reductase is inactivated-activated.     

Effect of liposome composition on the activity of detergent-solubilized acylcoenzyme A: cholesterol acyltransferase

Acylcoenzyme A:cholesterol acyltransferase (ACAT) was solubilized from Ehrlich ascites cell microsomes with Triton X-100. After removal of the detergent, ACAT activity per mg protein was reduced by 50 to 65% as compared with untreated microsomes. When this microsomal extract was combined with liposomes composed of cholesterol and egg phosphatidylcholine, the ACAT activity increased 5.4- to 6.7-fold. Under these conditions sucrose density gradient centrifugation indicated that more than 50% of the added lipid was incorporated into vesicles having the same density as the ACAT activity, suggesting the formation of a complex. ACAT activity increased 2.9-fold when the phosphatidylcholine content of the liposomes was raised from 0.5 to 5.0 mumol/mg microsomal protein. By contrast, the ACAT activity increased only 42% when the cholesterol content of the liposomes was raised from 0.17 to 0.57 mumol/mg microsomal protein. Addition of phosphatidylethanolamine to the liposomes produced little change in ACAT activity, whereas the activity was reduced by 25 and 50%, respectively, when sphingomyelin or phosphatidylserine was added. ACAT activity was five times higher when the liposomes were prepared from dioleoylphosphatidylcholine than from saturated phosphatidylcholines, including hydrogenated egg yolk, dimyristoyl or dipalmitoyl phosphatidylcholine. Likewise, the ACAT activity with liposomes made from soybean or egg yolk phosphatidylcholine was almost 3.5-fold greater than with those prepared from the saturated phosphatidylcholines. These results are consistent with the view that the activity of ACAT can be modified by changes in the composition of the membrane lipids with which the enzyme is associated.     

Protein metabolism in the tumour-bearing mouse. Rates of protein synthesis in host tissues and in an Ehrlich ascites tumour at different stages in tumour growth.

The activity of acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) was measured in fibroblast homogenates from Niemann-Pick Type C (NPC) and Type D (NPD) patients to determine whether these cells exhibit similar defects in the regulation of cholesterol esterification. ACAT activity in normal cells cultured in the absence of serum lipoproteins responded rapidly (within 6 h) to the addition of serum and reached peak levels at 12-24 h, whereas little stimulation of activity in NPC cells was observed. In contrast, ACAT activity in NPD fibroblasts (cell lines from four different patients) began to increase between 6 and 12 h after serum addition, reaching levels up to 50% of normal values at 24 h. ACAT activity in NPC and NPD cell extracts could not be stimulated by preincubation with normal cell homogenates, nor was complementation between NPC and NPD homogenates observed. Addition of 25-hydroxycholesterol to fibroblasts cultured in delipidated serum increased ACAT activity for all three cell types, although stimulation in NPD cells was less than that observed in NPC cells. ACAT activity of deoxycholate-solubilized homogenates reconstituted into phosphatidylcholine vesicles was independent of the presence of serum lipoproteins during culture and dependent on cholesterol present in the vesicles for all cell types. However, ACAT activities of mutant fibroblasts in vesicles plus cholesterol were significantly (about 40%) lower than control levels. These results suggest that the metabolic lesions in NPC and NPD cells are biochemically distinct and that both may involve factors in addition to the availability of cholesterol substrate for the ACAT enzyme.     

Changes in both acyl-CoA:cholesterol acyltransferase activity and microsomal lipid composition in rat liver induced by distal-small-bowel resection.

The acyl-CoA:cholesterol acyltransferase (ACAT) activity and lipid composition of hepatic microsomal membrane were investigated 6 weeks after both 50 and 75% distal-small-bowel resection (SBR). A significant decrease in hepatic cholesteryl ester levels was observed after SBR, with a significant increase in the cholesteryl ester content of the livers of 75% SBR compared with the 50% SBR. Hepatic total acylglycerols, free cholesterol and phospholipid levels were not modified after the surgical operation. Microsomal free cholesterol was increased after both 50 and 75% SBR. However, a decrease in both microsomal ACAT activity and cholesteryl ester levels were found in microsomes (microsomal fractions) of resected rats, both changes being higher after 75 than after 50% resection. The total phospholipid content of the microsomes did not change after the surgical operation. The microsomal phospholipid fatty acid composition indicated higher changes after 75 than after 50% SBR. These results demonstrated that, in resected animals: (1) the activity of the enzyme responsible for catalysing cholesterol esterification (ACAT) is decreased, and (2) hepatic microsomal free cholesterol does not appear to influence the activity of ACAT.     

Regulation of hepatic cholesterol ester hydrolase and acyl-coenzyme A:cholesterol acyltransferase in the rat

Cholesterol exists within the hepatocyte as free cholesterol and cholesteryl ester. The proportion of intrahepatic cholesterol in the free or ester forms is governed in part by the rate of cholesteryl ester formation by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolysis by neutral cholesterol ester (CE) hydrolase. In other cell types both ACAT and CE hydrolase activities are regulated in response to changes in the need for cellular free cholesterol. In rats, we performed a variety of experimental manipulations in order to vary the need for hepatic free cholesterol and to examine what effect, if any, this had on the enzymes that govern cholesteryl ester metabolism. Administration of a 20-mg bolus of lipoprotein cholesterol or a diet supplemented with 2% cholesterol resulted in an increase in microsomal cholesteryl ester content with little change in microsomal free cholesterol. This was accomplished by an increase in cholesteryl esterification as measured by ACAT but no change in CE hydrolase activity. An increased need for hepatic free cholesterol was experimentally induced by intravenous bile salt infusion or cholestyramine (3%) added to the diet. ACAT activity was decreased with both experimental manipulations compared to controls, while CE hydrolase activity did not change. Microsomal cholesteryl ester content decreased significantly with little change in microsomal free cholesterol content. Addition of exogenous liposomal cholesterol to liver microsomes from cholestyramine-fed and control rats resulted in a 784 +/- 38% increase in ACAT activity. Nevertheless, the decrease in ACAT activity with cholestyramine feeding was maintained. These studies allowed us to conclude that changes in hepatic free cholesterol needs are met in part by regulation of the rate of cholesterol esterification by ACAT without a change in the rate of cholesteryl ester hydrolysis by CE hydrolase.     

ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT

Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.     

Hepatic acyl-coenzyme A:cholesterol acyltransferase activity is decreased in patients with cholesterol gallstones

Altered hepatic cholesterol metabolism has been implicated in the etiology of cholesterol gallstones. This hypothesis has been examined by determining acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in liver biopsies from 31 cholesterol gallstone patients and 12 control subjects. Hepatic ACAT activity in gallstone patients was decreased to one-third that in controls (P less than 0.001). No differences in hepatic homogenate or microsomal free and total cholesterol concentrations were observed between the two groups. However, marked increases in free (107%) and total (98%) cholesterol concentrations were found in the cytosolic fraction of liver biopsies from gallstone patients. The total phospholipid concentration of the liver homogenate fraction was unchanged in both groups; however, the microsomal total phospholipid concentration was reduced by 17% (P less than 0.01) in gallstone samples compared with controls. This difference did not result in a significantly increased microsomal cholesterol/phospholipid ratio for the gallstone group (0.180 +/- 0.030) compared with the control group (0.169 +/- 0.042). These results show that hepatic ACAT activity is significantly decreased in cholesterol gallstone patients. These changes in ACAT activity in livers of patients with cholesterol gallstones are consistent with the known increase in the amount of free cholesterol secreted in the bile of these patients. Thus, the changes in ACAT activity may contribute to the pathogenesis of cholesterol gallstones.     

Incorporation of some glycoconjugates into the membrane of liposomes and interaction of ganglioside-containing liposomes with rat hepatocytes in vitro

Inclusion of some glycosides, gangliosides and ceruloplasmin into large (300-400 nm in diameter) unilamellar liposomes was performed. About 100% of the gangliosides, 30-50% of ceruloplasmin and 3-5% of the glycosides were incorporated into the phospholipid vesicles under these conditions. The liposomes containing ceruloplasmin or gangliosides, in contrast to the glycoside-containing vesicles, were precipitated in the presence of agglutinin from Ricinus communis. The interaction of phospholipid vesicles containing gangliosides with rat hepatocytes "in vitro" was studied. It was found that the incorporation of gangliosides into the liposomal membrane increased the liposomal lipid uptake by 50% as can be judged from the uptake of radioactive cholesterol. Possible mechanisms of incorporation of carbohydrate-containing compounds into liposomes are discussed. It is concluded that beside the density of carbohydrates the degree of their exposure on the liposomal membrane is important for specific interactions of the vesicles with lectins.     

Effect of distal small bowel resection on ACAT activity and microsomal lipid composition in rat small intestine

1. The acyl-CoA:cholesterol acyltransferase (ACAT) activity and lipid composition of intestinal microsomal membrane were investigated 6 weeks after both 50 and 75% distal small bowel resection (DSBR). 2. No changes in both microsomal ACAT activity and cholesteryl ester levels were found, while microsomal non-esterified cholesterol content was increased after the surgical operation. 3. The total phospholipid content of the microsomes did not change as a result of DSBR. 4. The microsomal phospholipid fatty acid composition showed a significant increase in saturated fatty acids together with no changes in both total monounsaturated and total polyunsaturated fatty acids after resection. 5. An increase in the levels of linoleic acid accompanied by a decrease in arachidonic acid was found in remnant intestine of resected rats.     

Transfer of cholesterol between liposomal membranes.

The transfer of cholesterol between liposomal membranes was examined. On incubation of liposomes compsoed of egg yolk phosphatidylcholine, phosphatidic acid and cholesterol (molar percentage, 65.8 : 1.3 : 32.9 or 65.5 : 6.3 : 31.2), almost complete equilibration of the cholesterol pools was achieved within 6 to 8 h at 37 degrees C. The rate of transfer of cholesterol from the liposomes, in which cholesterol was introduced by 'the exchange reaction', was not significantly different from that from liposomes prepared in the presence of cholesterol, in which the cholesterol was distributed homogenously. These findings indicate that half life for 'flip-flop' of cholesterol molecules in egg yolk phosphatidylcholine liposomes is less than 6 h at 37 degrees C. The transfer of cholesterol between liposomes was strongly dependent on temperature and was affected by the fatty acid composition of the phospholipid, suggesting that the 'fluidity' of the membranes strongly influences the transfer rate. A preferential distribution of cholesterol molecules was observed in heterogeneous liposomes with different classes of phospholipids. The 'affinity order' of cholesterol for phospholipid deduced from the present experiments is as follows: beef brain sphingomyelin greater than dipalmitoylglycerophosphocholine = dimyristoylglycerophosphocholine greater than egg yolk phosphatidylcholine.     

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Effects of chylomicron remnants and beta-VLDL on the class and composition of newly secreted lipoproteins by HepG2 cells

The regulation of lipoprotein secretion in the cell line HepG2 was studied. HepG2 cells were preincubated with chylomicron remnants (triglyceride- and cholesterol-rich) or with beta very low density lipoproteins (beta-VLDL) (cholesterol-rich). The medium was removed and the cells were incubated for and additional 24 hr in a lipoprotein-free medium that contained either [2-3H]glycerol or DL-[2-3H]mevalonate. Cells and media were harvested, and lipoproteins were separated and fractionated. The mass and radioactivity of the lipids in cells and in the lipoproteins were measured. The activities of cellular acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase were also determined. Preincubation with chylomicron remnants induced an increase in cellular triglyceride and stimulated both HMG-CoA reductase and ACAT. Preincubation with beta-VLDL induced an increase in cellular free and esterified cholesterol, inhibited HMG-CoA reductase and stimulated ACAT. Although the absolute amount of VLDL is small, chylomicron remnants induced large relative increases in the amount of triglyceride and phospholipid secreted in VLDL and decreases in the amount of triglyceride secreted in low density (LDL) and high density (HDL) lipoproteins as well as a decrease in the amount of phospholipid secreted in HDL. In contrast, preincubation with beta-VLDL did not affect triglyceride secretion, but markedly stimulated the amount of phospholipid secreted in HDL. Comparison of the mass of glycerolipid actually secreted with that calculated from the cellular specific activity suggested that glycerolipids are secreted from single, rapidly equilibrating pools. Cholesterol and cholesteryl ester secretion were affected differently. Preincubation with chylomicron remnants increased the amount of free cholesterol secreted in both VLDL and LDL, but did not alter cholesteryl ester secretion. Preincubation with beta-VLDL increased free cholesterol secretion in all lipoprotein fractions and increased cholesteryl ester secretion in VLDL and LDL, but not HDL. Comparison of isotope and mass data suggested that the cholesteryl ester secreted came primarily from a preformed, rather than an newly synthesized, pool. In summary, these data provide insight to the mechanism whereby a liver cell regulates the deposition of exogenous lipid.     

Disintegration of phosphatidylcholine liposomes in plasma as a result of interaction with high-density lipoproteins.

1. During in vitro incubation of liposomes or unilamellar vesicles prepared from egg-yolk or rat-liver phosphatidylcholine with human, monkey or rat plasma the phospholipid becomes associated with a high molecular weight protein-containing component. 2. The phosphatidylcholine . protein complex thus formed co-chromatographs with high-density lipoprotein on Ultrogel AcA34 and has the same immunoelectrophoretic properties as this lipoprotein. 3. Release of the phosphatidylcholine from liposomes was also observed when liposomes were incubated with pure monkey high-density lipoproteins. Under those conditions some transfer of protein from the lipoprotein to the liposomes was observed as well. 4. The observed release of phospholipid from the liposomes is a one-way process, as the specific radioactivity of liposome-associated phosphatidylcholine remained constant during incubation with plasma. 5. It is concluded that either the lipoprotein particle takes up additional phospholipid or that a new complex is formed from protein constituents of the lipoprotein and the liposomal phosphatidylcholine. 6. Massive release of entrapped 125I-labeled albumin from the liposome during incubation with plasma suggests that the observed release of phosphatidylcholine from the liposomes has a highly destructive influence on the liposomal structure. 7. Our results are discussed with special reference to the use of liposomes as intravenous carriers of drugs and enzymes.