Interaction of lipoproteins with type II pneumocytes in vitro: morphological studies, uptake kinetics and secretion rate of cholesterol.
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To examine the role that lipoprotein charge plays in cholesterol metabolism in vivo, we characterized the effects of an intravenous injection of 40 μmol of an uncharged phospholipid (phosphatidylcholine, PC) or an anionic phospholipid (phosphatidylinositol, PI) into fasted rabbits. PC injection had a negligible effect on lipoprotein charge and composition, similar to that observed in a saline-injected animal. In contrast, PI injection caused a significant increase in the net negative surface charge of all lipoproteins after only 10 min, followed by a gradual return to normal by 24 h. Lipoprotein compositional analysis showed that PI caused a significant increase of cholesteryl ester (CE) and cholesterol (FC) in the VLDL pool by 3 h, with no changes in VLDL-triglyceride content. While the bulk of the plasma CE was located in the HDL pool in the PC-injected animals, in the PI animals, VLDL became the major CE storage compartment. No major changes in the levels or composition of HDL or LDL were evident over the 24-h turnover period. Co-injection of [3H]FC revealed a 30-fold greater rate of clearance of the labeled cholesterol from the PI-injected rabbit plasma. In addition, the rate of cholesterol esterification by lecithin:cholesterol acyltransferase was almost completely inhibited in the PI animals. In summary, a bolus injection of PI into rabbits appears to enhance the mobilization of cellular sterol and promote a rapid clearance of both FC and CE from the plasma compartment. The data show that lipoprotein charge can affect cholesterol transport and that this process can be selectively manipulated. —Stamler, C. J., D. Breznan, T. A-M. Neville, F. J. Viau, E. Camlioglu, and D. L. Sparks. Phosphatidylinositol promotes cholesterol transport in vivo. J. Lipid Res. 2000. 41: 1214–1221. To examine the role that lipoprotein charge plays in cholesterol metabolism in vivo, we characterized the effects of an intravenous injection of 40 μmol of an uncharged phospholipid (phosphatidylcholine, PC) or an anionic phospholipid (phosphatidylinositol, PI) into fasted rabbits. PC injection had a negligible effect on lipoprotein charge and composition, similar to that observed in a saline-injected animal. In contrast, PI injection caused a significant increase in the net negative surface charge of all lipoproteins after only 10 min, followed by a gradual return to normal by 24 h. Lipoprotein compositional analysis showed that PI caused a significant increase of cholesteryl ester (CE) and cholesterol (FC) in the VLDL pool by 3 h, with no changes in VLDL-triglyceride content. While the bulk of the plasma CE was located in the HDL pool in the PC-injected animals, in the PI animals, VLDL became the major CE storage compartment. No major changes in the levels or composition of HDL or LDL were evident over the 24-h turnover period. Co-injection of [3H]FC revealed a 30-fold greater rate of clearance of the labeled cholesterol from the PI-injected rabbit plasma. In addition, the rate of cholesterol esterification by lecithin:cholesterol acyltransferase was almost completely inhibited in the PI animals. In summary, a bolus injection of PI into rabbits appears to enhance the mobilization of cellular sterol and promote a rapid clearance of both FC and CE from the plasma compartment. The data show that lipoprotein charge can affect cholesterol transport and that this process can be selectively manipulated. —Stamler, C. J., D. Breznan, T. A-M. Neville, F. J. Viau, E. Camlioglu, and D. L. Sparks. Phosphatidylinositol promotes cholesterol transport in vivo. J. Lipid Res. 2000. 41: 1214–1221. The factors that regulate cholesterol flux to the liver are as yet poorly understood but are thought to involve two distinct systems: a cellular sterol regulatory system and an intravascular transport system. The classic view has been that excess extrahepatic cholesterol can be transported in high density lipoprotein (HDL) particles to the liver for excretion (1Glomset J.A. The plasma lecithin:cholesterol acyltransferase reaction. (Review).J. Lipid Res. 1968; 9: 155-167Google Scholar). HDL has been shown to be able to adsorb cholesterol and cholesteryl esters (CE) from cell membranes (2Phillips M.C. Gillotte K.L. Haynes M.P. Johnson W.J. Lund-Katz S. Rothblat G.H. Mechanisms of high density lipoprotein-mediated efflux of cholesterol from cell plasma membranes.Atherosclerosis. 1998; 137: S13-S17Google Scholar, 3de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar, 4Graf G.A. Connell P.M. van der Westhuyzen D.R. Smart E.J. The class B, type I scavenger receptor promotes the selective uptake of high density lipoprotein cholesterol ethers into caveolae.J. Biol. Chem. 1999; 274: 12043-12048Google Scholar). Studies suggest that this efflux of sterol may be closely regulated by the cell and may involve specific cell surface domains called caveolae (5Fielding P.E. Fielding C.J. Plasma membrane caveolae mediate the efflux of cellular free cholesterol.Biochemistry. 1995; 34: 14288-14292Google Scholar). HDL is thought to transport sterol either directly or indirectly to the liver and clearance appears to involve specific cell-surface receptors called scavenger receptor-BI (SR-BI) (6Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor (see comments).Science. 1996; 271: 518-520Google Scholar). The direct pathway describes the undeviating delivery of this lipid to the liver, which is thought to account for approximately 30% of hepatic uptake of CE in rabbits (7Goldberg D.I. Beltz W.F. Pittman R.C. Evaluation of pathways for the cellular uptake of high density lipoprotein cholesterol esters in rabbits.J. Clin. Invest. 1991; 87: 331-346Google Scholar). The second, indirect, pathway constitutes the major hepatic sterol uptake route in rabbits (7Goldberg D.I. Beltz W.F. Pittman R.C. Evaluation of pathways for the cellular uptake of high density lipoprotein cholesterol esters in rabbits.J. Clin. Invest. 1991; 87: 331-346Google Scholar). The sterol is initially transferred from HDL to the rapidly turning over VLDL remnant lipoprotein pool, from which it is then cleared by the liver (8Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Google Scholar, 9Tall A.R. An overview of reverse cholesterol transport.Eur. Heart J. 1998; 19: A31-A35Google Scholar). Intravascular sterol transport is thought to primarily involve the actions of two different plasma proteins: lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP). The consensus view has been that LCAT may form a concentration gradient to move sterol into and through the plasma compartment by promoting the conversion of free cholesterol (FC) to CE on HDL particles (10Jonas A. Lecithin:cholesterol acyltransferase.in: Gotto Jr., A.M. Plasma Lipoproteins. Elsevier, Amsterdam1987: 299-333Google Scholar). CETP may then promote this lipid flux by moving the newly formed CE from HDL to the apoB-containing lipoprotein pool (11Tall A.R. Plasma lipid transfer proteins.J. Lipid Res. 1986; 27: 361-367Google Scholar, 12Lagrost L. The role of cholesteryl ester transfer protein and phospholipid transfer protein in the remodeling of plasma high-density lipoproteins.Trends Cardiovasc. Med. 1997; 7: 218-224Google Scholar). Studies have shown that both the rate of FC esterification and the transfer of these CE into VLDL are enhanced, both in a postprandial lipemic state (13Francone O.L. Gurakar A. Fielding C. Distribution and functions of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in plasma lipoproteins. Evidence for a functional unit containing these activities together with apolipoproteins A-I and D that catalyzes the esterification and transfer of cell-derived cholesterol.J. Biol. Chem. 1989; 264: 7066-7072Google Scholar, 14Mowri H.O. Patsch J.R. Ritsch A. Fwöger B. Brown S. Patsch W. High density lipoproteins with differing apolipoproteins: Relationships to postprandial lipemia, cholesteryl ester transfer protein, and activities of lipoprotein lipase, hepatic lipase, and lecithin:cholesterol acyltransferase.J. Lipid Res. 1994; 35: 291-300Google Scholar) and also in some hyperlipidemic states (12Lagrost L. The role of cholesteryl ester transfer protein and phospholipid transfer protein in the remodeling of plasma high-density lipoproteins.Trends Cardiovasc. Med. 1997; 7: 218-224Google Scholar, 15Tall A. Granot E. Brocia R. Tabas I. Hesler C. Williams K. Denke M. Accelerated transfer of cholesteryl esters in dyslipidemic plasma. Role of cholesteryl ester transfer protein.J. Clin. Invest. 1987; 79: 1217-1225Google Scholar, 16Dobiasova M. Stribrna J. Sparks D.L. Pritchard P.H. Frohlich J.J. Cholesterol esterification rates in very low density lipoprotein- and low density lipoprotein-depleted plasma. Relation to high density lipoprotein subspecies, sex, hyperlipidemia, and coronary artery disease.Arterioscler. Thromb. 1991; 11: 64-70Google Scholar). This CETP-mediated enhanced transfer of CE into VLDL was originally thought to be a result of a reciprocal mass exchange of CE and triglyceride between HDL and VLDL (11Tall A.R. Plasma lipid transfer proteins.J. Lipid Res. 1986; 27: 361-367Google Scholar). More recent work has shown, however, that both the presence of preferred triglyceride (TG)-enriched substrates and end products of lipolysis can influence the actions of CETP and enhance transfer into VLDL (12Lagrost L. The role of cholesteryl ester transfer protein and phospholipid transfer protein in the remodeling of plasma high-density lipoproteins.Trends Cardiovasc. Med. 1997; 7: 218-224Google Scholar). Other work has revealed that the electrostatic properties of plasma lipoproteins can also affect the interlipoprotein transfer of lipid by CETP (17Nishida H.I. Arai H. Nishida T. Cholesterol ester transfer mediated by lipid transfer protein as influenced by changes in the charge characteristics of plasma lipoproteins.J. Biol. Chem. 1993; 268: 16352-16360Google Scholar, 18Masson D. Athias A. Lagrost L. Evidence for electronegativity of plasma high density lipoprotein-3 as one major determinant of human cholesteryl ester transfer protein activity.J. Lipid Res. 1996; 37: 1579-1590Google Scholar). Investigations with LCAT have shown that this enzyme is also sensitive to charge properties of HDL (19Jonas A. Daehler J.L. Wilson E.R. Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines. apolipoprotein A-I cholesterol.Biochim. Biophys. Acta. 1986; 876: 474-485Google Scholar, 20Sparks D.L. Frank P.G. Neville T.A. Effect of the surface lipid composition of reconstituted LPA-I on apolipoprotein A-I structure and lecithin:cholesterol acyltransferase activity.Biochim. Biophys. Acta. 1998; 1390: 160-172Google Scholar). While an increase in the protein-dependent negative charge on HDL was shown to be associated with a stimulation of LCAT, increased anionic lipid content in HDL had an inhibitory effect on the enzyme (20Sparks D.L. Frank P.G. Neville T.A. Effect of the surface lipid composition of reconstituted LPA-I on apolipoprotein A-I structure and lecithin:cholesterol acyltransferase activity.Biochim. Biophys. Acta. 1998; 1390: 160-172Google Scholar). All lipoprotein classes exhibit a net negative charge and studies have shown that this charge is due to both the apolipoprotein composition of the lipoprotein and its content of anionic lipids (specifically phosphatidylinositol (PI)) (21Sparks D.L. Phillips M.C. Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.J. Lipid Res. 1992; 33: 123-130Google Scholar, 22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar). Experiments in this laboratory have shown that interfacially active enzymes are affected by lipoprotein charge and that HDL metabolism in vivo is directly affected by the charge on HDL particles (23Braschi S. Neville T.A. Vohl M.C. Sparks D.L. Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo.J. Lipid Res. 1999; 40: 522-532Google Scholar). The data suggest that lipoprotein charge may affect cholesterol metabolism in vivo. The present study examines the effects of the anionic lipid, PI, on lipoprotein charge and cholesterol metabolism in a rabbit. We show that this lipid: 1) increases the negative surface charge of all the plasma lipoproteins, 2) increases VLDL cholesterol content, and 3) stimulates a sterol flux through the plasma compartment. The study shows that cholesterol metabolism in vivo can be manipulated by altering the charge characteristics of plasma lipoproteins. [3H]cholesterol was purchased from Mandel NEN Life Science Producers (Guelph, ON). 1-Palmitoyl-2-oleoyl phosphatidylcholine (PC) and phosphatidylinositol (PI) were obtained from Avanti Polar Lipids (Birmingham, AL). Silica Gel Impregnated Glass Fiber Sheets (ITLC™ SG) were obtained from Gelman Sciences (Ann Arbor, MI) and silica gel 60 plates were obtained from EM Science (Gibbstown, NJ). All other reagents were of analytical grade. PI and PC vesicles were prepared by drying 40 μmol of each lipid to completion in a 12 × 75 mm culture tube under N2. The lipids were solubilized in 3 mL of 50 mm sodium phosphate, pH 7.2, 150 mm sodium chloride (PBS) by sonication for 1 min at constant duty cycle. The vesicles were incubated at 37°C for 10 min and then sonicated at a high output for 4 min in 10°C water bath under N2. Samples were centrifuged for 5 min at 3,000 rpm to remove any particulate titanium. Male New Zealand white rabbits (3.5–4.0 kg) were fasted for 12 h prior to the experiment and remained fasted until after the final time point sample was taken. Rabbits had free access to water during this time. A catheter was inserted into the marginal ear vein and blood samples were collected into tubes containing 7.5% (K3) EDTA solution at the desired time points. A pre-injection blood sample was taken and the vesicle solution of either PI (n = 4), or PC (n = 2) or saline (n = 1) was injected into the marginal ear vein. A sample of blood was removed at 10 and 30 min, 1, 3, 6, and 24 h after the injection of the lipid vesicles. All blood samples were placed on ice and then centrifuged at 3,000 rpm for 15 min at 4°C to separate the plasma. In order to ensure that LCAT was inhibited in the stored plasma, iodoacetamide (150 mm) was added to plasma samples (24Guerin M. Dolphin P.J. Chapman M.J. A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies. Identification of preferential cholesteryl ester acceptors in human plasma.Arterioscler. Thromb. 1994; 14: 199-206Google Scholar). In order to determine the rate of clearance of cholesterol from the rabbit, a radioactive tracer was added to the vesicle preparations prior to injection into the rabbit. Two hundred μCi [3H]FC was dried in a 12 × 75 mm culture tube with 40 μmol of PI or POPC. Three ml of PBS was added to the dried lipids and the mixture was sonicated as described above. In some studies, to verify that PI did not affect the incorporation/clearance of the tracer, the tracer was combined with 1 mg of PC and 3 mL of PBS and vesicles were prepared as described above. This tracer/vesicle preparation was injected and then, 5 min later, the PI or PC vesicles were injected and blood was sampled as described. Lipoprotein fractions were isolated by sequential ultracentrifugation (VLDL+IDL, d < 1.019 g/mL; LDL, d 1.019–1.063 g/mL; and HDL, d 1.063–1.21 g/mL) and lipoprotein lipid composition (total cholesterol, FC, and TG concentrations) was determined enzymatically using kits from Roche Diagnostic (Laval, PQ). An aliquot of each lipoprotein was dialyzed into PBS and surface charge characteristics were determined by electrophoresis on pre-cast 0.5% agarose gels (Beckman, Paragon Lipo Kit) (21Sparks D.L. Phillips M.C. Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.J. Lipid Res. 1992; 33: 123-130Google Scholar). Protein concentrations were determined using the Lowry method as modified by Markwell et al. (25Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). PI concentration was determined after lipid extraction in chloroform and methanol by high performance thin-layer chromatography on silica gel 60 plates using a solvent system composed of chloroform–methanol–ammonia, 65:35:5 (v/v/v). Lipids were charred with a 10% copper sulfate (w/v), 8% phosphoric acid (w/w) solution and PI concentration was quantified from a standard curve. To determine the amount of 3H associated with FC and CE, a fraction of each lipoprotein (20 μL) and whole plasma (40 μL) was extracted in ethanol and hexane and the lipids were separated by thin-layer chromatography on ITLC™ SG plates using a solvent system composed of hexane–diethyl ether–acetic acid, 90:10:1 (v/v/v). The effect of PI on LCAT activity was examined in incubations of plasma samples that were not treated with iodoacetamide. Four hundred μL of plasma at each time point was incubated for 30 min with 10 μCi of [3H]FC on filter paper discs (16Dobiasova M. Stribrna J. Sparks D.L. Pritchard P.H. Frohlich J.J. Cholesterol esterification rates in very low density lipoprotein- and low density lipoprotein-depleted plasma. Relation to high density lipoprotein subspecies, sex, hyperlipidemia, and coronary artery disease.Arterioscler. Thromb. 1991; 11: 64-70Google Scholar) at 37°C and the reaction was terminated with the addition of 2 mL ethanol. Reaction products were extracted in hexane and the amount of 3H associated with CE and FC was determined by thin-layer chromatography as described above. Figure 1 illustrates the effect of injection of PI and PC vesicles on the estimated surface potential of HDL, LDL, and VLDL. While injection of PC or saline (not shown) had no significant effect on lipoprotein charge, injection of PI vesicles caused all lipoprotein fractions to migrate further on the agarose gel, indicative of an increased negative surface charge. This increased negative charge inlipoproteins from the PI-injected rabbit reached a peak early in the time course and then returned to normal by 24 h. The HDL fraction exhibited a background surface potential of −12.2 mV, prior to the PI injection and then reached a peak negative charge of −18.0 mV 10 min after the PI injection. Similarly, the VLDL fraction had an initial surface potential of −8.8 mV, which increased to −11.2 mV after 10 min. LDL charge peaked somewhat later; LDL had an initial surface potential of −3.9 mV, and peaked 60 min after the PI injection at −8.0 mV. Plasma lipoprotein compositions of ultracentrifugally isolated lipoproteins sampled before and after the bolus injection of phospholipid were determined. The protein concentrations of the HDL fractions remained relatively constant after injection of the different lipids, while the protein concentrations in the LDL and VLDL fractions increased slightly in both the PC and PI injected animals over time (Table 1). The HDL and LDL fractions exhibited no significant change in either TG/protein, CE/protein, or FC/protein ratios after injection of PI. However, injection of PC or saline was associated with a small reduction in HDL CE and FC and a concomitant increase in these lipids in LDL (Table 1). In PI-injected animals, HDL and LDL-PI concentrations increased from being below detection levels to 33.3 and 3.6 nmol/ml plasma, respectively, by 10 min post injection. The PI concentration in VLDL increased to 1.4 nmol/ml by 30 min and then all PI concentrations decreased to undetectable background levels by 3 h. VLDL fractions from the PI-injected rabbit showed a significant change in FC/protein and CE/protein ratios (Fig. 2). While the FC/protein ratios in VLDL increased after both PC and PI injections, the magnitude of increase was 3-fold greater in the PI-injected animal and peaked 3 h post injection (Fig. 2A). Figure 2B shows a similar 3-fold increase in VLDL-CE, which also peaked 3 h after the injection. These increases in VLDL-CE shifted the relative plasma CE storage location from the HDL pool to the VLDL pool (65–30% and 10–50% of total plasma CE, respectively). This was followed by a subsequent return to near normal VLDL compositional values by 24 h. No other changes in VLDL composition were observed in PI-injected rabbits and there was only a slight increase in CE and TG in the VLDL pools after injection of PC or saline.TABLE 1.Effect of phospholipid injection on lipoprotein lipid and protein concentrationsCE/ProteinFC/ProteinTG/ProteinProteinmg/mg proteinmg/dlPC InjectionHDLt = 00.23 ± 0.020.06 ± 0.010.12 ± 0.0157.4 ± 2.2t = 30.14 ± 0.020.04 ± 0.010.17 ± 0.0243.9 ± 0.7t = 240.11 ± 0.010.04 ± 0.010.20 ± 0.0146.9 ± 1.7LDLt = 03.1 ± 0.80.41 ± 0.052.7 ± 0.21.7 ± 0.1t = 39.2 ± 0.50.87 ± 0.103.3 ± 0.31.4 ± 0.1t = 245.8 ± 0.30.33 ± 0.072.6 ± 0.23.1 ± 0.1VLDLt = 00.22 ± 0.020.03 ± 0.011.6 ± 0.15.7 ± 0.3t = 30.44 ± 0.080.23 ± 0.023.4 ± 0.16.3 ± 0.1t = 240.53 ± 0.050.32 ± 0.013.8 ± 0.29.4 ± 0.2PI InjectionHDLt = 00.63 ± 0.070.13 ± 0.010.29 ± 0.0326.6 ± 2.4t = 30.63 ± 0.120.15 ± 0.010.36 ± 0.0531.8 ± 3.3t = 240.70 ± 0.060.16 ± 0.030.42 ± 0.0629.3 ± 2.6LDLt = 01.8 ± 0.30.43 ± 0.021.6 ± 0.33.5 ± 0.1t = 31.8 ± 0.30.47 ± 0.031.7 ± 0.25.3 ± 0.2t = 242.0 ± 0.20.49 ± 0.032.0 ± 0.18.8 ± 0.5VLDLt = 00.34 ± 0.040.03 ± 0.023.5 ± 0.22.9 ± 0.1t = 31.50 ± 0.30.79 ± 0.064.4 ± 0.22.6 ± 0.1t = 240.70 ± 0.20.50 ± 0.042.3 ± 0.25.4 ± 0.2Phosphatidylinositol (PI) and phosphatidylcholine (PC) vesicles (40 μmol) were injected into fasting rabbits. Plasma was sampled at various times (t) and lipoproteins were isolated ultracentrifugally. Lipoprotein triglyceride (TG) cholesterol (FC), and cholesteryl ester (CE) concentrations (mg/mg protein) were determined as described and are representative of two injections of PC and four injections of PI in rabbits. Protein concentrations (mg/dl) were determined as described and values are representative of two injections of PC and four injections of PI in rabbits. Open table in a new tab Phosphatidylinositol (PI) and phosphatidylcholine (PC) vesicles (40 μmol) were injected into fasting rabbits. Plasma was sampled at various times (t) and lipoproteins were isolated ultracentrifugally. Lipoprotein triglyceride (TG) cholesterol (FC), and cholesteryl ester (CE) concentrations (mg/mg protein) were determined as described and are representative of two injections of PC and four injections of PI in rabbits. Protein concentrations (mg/dl) were determined as described and values are representative of two injections of PC and four injections of PI in rabbits. Figure 3 compares the clearance of [3H]FC at each time point for rabbits injected with PI or PC vesicles. It is evident that the radioactive FC in plasma rapidly fell to approximately 15% of the initial dose after only 1 h in the rabbit injected with PI. In comparison, the rabbit injected with PC still contained approximately 90% of the injected radioactivity after 1 h. The PC-injected rabbit took more than 6 h to clear [3H]FC to a baseline level comparable to that of an animal injected with PI. The half-life of [3H]FC in the PI-injected rabbits (0.3 h) was approximately 30-fold shorter than that for the PC-injected rabbits (8.4 h). To determine whether the tracer clearance was affected by co-injection of [3H]FC with different lipids, experimentswere undertaken where [3H]FC was complexed with a small amount (1 mg) of PC, injected into the rabbit, and then PI vesicles were injected 5 min later. This resulted in the same rapid clearance of the tracer as was seen with the co-injection of PI and [3H]FC (data not shown). Figure 4 shows the amount of newly formed [3H]CE remaining in the plasma after the injection of [3H]FC incorporated into either PC or PI vesicles. Initial 10-min levels of [3H]CE were >6 times lower in the PI-injected rabbit, as compared to the PC-injected rabbit. The levels of [3H]CE in the PI-injected rabbit began to rise after 1 h and eventually rose 4-fold by 24 h. In the PC-injected rabbit, the level of [3H]CE fell to approximately 10% of the injected dose by 6 h and remained constant until 24 h. It is notable that after 24 h, there was almost twice the amount of [3H]CE in the plasma of the PI-injected rabbit, relative to that of the PC-injected animal. Figure 5A and B illustrates the distribution of [3H]FC and [3H]CE in each lipoprotein fraction. In the PC-injected rabbit, [3H]FC was predominantly found in the LDL and HDL fractions at the early time points (Fig. 5A). By 6 h, the amount of [3H]FC in LDL and HDL fell and paralleled a major increase in the VLDL [3H]FC content. In contrast, in the PI-injected animal, most of the [3H]FCwas found in the VLDL fraction at the early time points, while by 24 h, [3H]FC was approximately equally distributed through all the lipoprotein fractions. Newly synthesized [3H]CE distribution in the different lipoprotein fractions showed quite different patterns in the PI- and PC-injected animals (Fig. 5B). In the PC-injected rabbits, [3H]CE was enriched in the HDL pool and depleted in the VLDL pool. However, in the PI-injected rabbits, [3H]CE levels in HDL were reduced by almost 50% relative to the PC animals, and paralleled an equivalent increase of [3H]CE in the VLDL pool. The effect of PC or PI injections on the rate of cholesterol esterification by LCAT was measured in individual plasma samples from the various time points. No major change in LCAT activity was observed after injection of PC vesicles (Fig. 6). In contrast, Fig. 6 shows that endogenous LCAT activity was significantly inhibited (18% of normal) 10 min after injection of PI. PI injection reduced the fractional rates of cholesterol esterification from 45% to 8%per hour. Six hours after the PI vesicle injection, LCAT activity returned to approximately 75% of normal. Studies have shown that alterations in the composition of lipoprotein particles can affect their charge and structural properties and that these characteristics will influencelipoprotein remodelling and plasma lipid metabolism (26Patsch J.R. Prasad S. Gotto Jr., A.M. Patsch W. High density lipoprotein2. Relationship of the plasma levels of this lipoprotein species to its composition, to the magnitude of postprandial lipemia, and to the activities of lipoprotein lipase and hepatic lipase.J. Clin. Invest. 1987; 80: 341-347Google Scholar, 27De Rijke Y.B. Biessen E.A.L. Vogelezang C.J.M. Van Berkel T.J.C. Binding characteristics of scavenger receptors on liver endothelial and Kupffer cells for modified low-density lipoproteins.Biochem. J. 1994; 304: 69-73Google Scholar, 28La Belle M. Blanche P.J. Krauss R.M. Charge properties of low density lipoprotein subclasses.J. Lipid Res. 1997; 38: 690-700Google Scholar). In general, lipoprotein charge is primarily due to the specific apolipoprotein composition of the lipoprotein class (22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar, 29Chappey B. Myara I. Benoit M.O. Mazière C. Mazière J.C. Moatti N. Characteristics of ten charge-differing subfractions isolated from human native low-density lipoproteins (LDL). No evidence of peroxidative modifications.Biochim. Biophys. Acta. 1995; 1259: 261-270Google Scholar, 30Chauhan V. Wang X. Ramsamy T. Milne R.W. Sparks D.L. Evidence for lipid-dependent structural changes in specific domains of apolipoprotein B100.Biochemistry. 1998; 37: 3735-3742Google Scholar). Our studies have shown that this protein-dependent charge is governed by the conformation of the apolipoprotein and therefore directly affected by different uncharged lipids that are able to modify the protein conformation (31Sparks D.L. Lund-Katz S. Phillips M.C. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.J. Biol. Chem. 1992; 267: 25839-25847Google Scholar, 32Sparks D.L. Davidson W.S. Lund-Katz S. Phillips M.C. Effects of the neutral lipid content of high density lipoprotein on apolipoprotein A-I structure and particle stability.J. Biol. Chem. 1995; 270: 26910-26917Google Scholar). In addition, lipoprotein charge is also affected by its content of charged molecules, predominantly anionic lipids such as PI and non-esterified free fatty acids (NEFA) (22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar). PI is an anionic lipid found in all classes of lipoproteins and accounts for approximately 4% of the total phospholipid (PL) in HDL (22Davidson W.S. Sparks D.L. Lund-Katz S. Phillips M.C. The molecular basis for the difference in charge between pre-beta- and alpha-migrating high density lipoproteins.J. Biol. Chem. 1994; 269: 8959-8965Google Scholar). Little is known of what affects or regulates the amount of PI in the different lipoprotein classes. In contrast, numerous factors can affect
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Mouse peritoneal macrophages were incubated with abnormal lipoproteins (LP-X, HDL-E, VLDL-p, IDl-p and LDL-p) from a patient with secondary deficiency in phosphatidylcholine-sterol acyltransferase, or with phosphatidylcholine/cholesterol liposomes, and the stimulation of cholesteryl ester formation was studied. Acetylated low density lipoproteins served as a control. It was found that macrophages incubated with LP-X, the other pathological lipoproteins or with liposomes did not show an enhanced cholesterol esterification. Also HDL-E had no effect despite of its high apoE content and the fact that apoE has been postulated to be the agonist in beta-VLDL binding to macrophages.
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We have examined cholesteryl ester transfer (CET) from HDL to low density and very low density lipoproteins (LDL and VLDL) and lecithin: cholesterol acyl transferase (LCAT) activity in plasma from 28 men with non-insulin-dependent diabetes mellitus (NIDDM) treated with diet alone or diet and sulphonylurea drugs and in 27 healthy non-diabetic controls. Patients and healthy subjects had similar LCAT activity, but CET was significantly higher in NIDDM 26.1 +/- 11.5 mumol l-1 h-1) than in healthy men (17.8 +/- 6.5 mumol l-1 h-1) (p = 0.001). Diabetic men also had higher CET compared to 15 healthy non-diabetic men (18.7 +/- 5.6 mumol l-1 h-1) (p = 0.001) with similar serum lipids. CET activity was similar in patients treated with diet alone (24.8 +/- mumol l-1 h-1) or with sulphonylureas (27.7 +/- 15.8 mumol l-1 h-1). The Sf 0-12 fraction was significantly enriched with total cholesterol (p = 0.0001) and free cholesterol (p = 0.0006) in diabetic subjects whether treated with diet alone or on sulphonylureas compared to the 15 non-diabetic controls matched for serum triglycerides. The free cholesterol/phospholipid, the free cholesterol/total protein and the free cholesterol/mass ratios were increased in the Sf 0-12 fraction in diabetic subjects (p < 0.01). These findings indicate that CET is accelerated in patients with NIDDM and that this may be due to the altered composition of acceptor lipoproteins.
Cholesterylester transfer protein
Sterol O-acyltransferase
Phospholipid transfer protein
High-density lipoprotein
Cholesteryl ester
Reverse cholesterol transport
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Citations (34)
Abstract Male adult Wistar rats received daily (at 9 a.m. and 5 p.m.) 10 μg of zinc‐protamine glucagon by subcutaneous injection for 8 days. Plasma cholesterol levels were decreased by 36% in fed rats, 33% in cholesterol‐fed rats and by 55% in fasted rats. Lipoproteins were separated into 22 fractions by ultracentrifugation using a density gradient. Glucagon administration decreased the cholesterol content in all lipoproteins except low density lipoprotein (LDL 1 ) (1.006–1.040) and very low density lipoprotein (VLDL) from cholesterol‐fed rats. The main decrease (−57 to −81%) was observed in 1.050–1.100 g/mL lipoproteins (LDL 2 and HDL 2 ), which contained a large amount of apo E, while HDL 3 cholesterol was not affected. Triacylglycerol levels were decreased only in chylomicrons and VLDL (−70%) of fed and cholesterol‐fed rats, while plasma and lipoprotein triacylglycerol levels were not changed in fasted rats treated with glucagon. In normally fed rats glucagon administration increased by 42% the fractional catabolic rate of [ 125 I]HDL 2 while the absolute catabolic rate appeared to be unchanged. Glucagon seems to be a potent hypolipidemic agent affecting mainly the apo E‐rich lipoproteins. Its chronic administration limits lipoprotein accumulation which occurs upon cholesterol feeding.
Lipidology
Chylomicron
Catabolism
High-density lipoprotein
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Citations (27)
The effects of dietary eritadenine on the concentration of plasma lipoprotein lipids and the molecular species profile of plasma lipoprotein phosphatidylcholine (PC) were investigated in rats fed cholesterol-free and cholesterol-enriched diets to obtain insights into the relationship between the changes in PC molecular species profile and the hypocholesterolemic action of eritadenine. The effect of eritadenine on the secretion rate of very low density lipoprotein (VLDL) from the liver was also estimated. Rats were fed the control or eritadenine-supplemented (50 mg/kg) diets with or without exogenous cholesterol for 14 d. Eritadenine supplementation significantly decreased the cholesterol of major plasma lipoproteins, high density lipoprotein and VLDL, in rats fed cholesterol-free and cholesterol-enriched diets, respectively. The ratio of PC to phosphatidylethanolamine, delta6-desaturase activity, and the ratio of arachidonic acid to linoleic acid in liver microsomes were markedly decreased by eritadenine irrespective of the presence or absence of exogenous cholesterol. Dietary eritadenine increased the proportion of 16:0-18:2 molecular species with a decrease in 18:0-20:4 in plasma lipoprotein PC in both rats fed cholesterol-free and cholesterol-enriched diets. Eritadenine did not depress the secretion rate of VLDL in rats fed a cholesterol-free diet containing a high level of choline. The results indicate that dietary eritadenine elicits its hypocholesterolemic action with modulations of the fatty acid and molecular species profiles of PC irrespective of the presence or absence of exogenous cholesterol. The eritadenine-induced alteration of PC molecular species profile is discussed in relation to the hypocholesterolemic action of eritadenine.
Plasma lipoprotein
Intermediate-density lipoprotein
Reverse cholesterol transport
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Citations (36)
We have investigated the transfer of [14C]cholesterol from labeled bovine heart mitochondria and Friend erythroleukemic cells to high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions from human and rat plasma. The lipoprotein fractions were obtained by molecular sieve chromatography of plasma on agarose A-5m columns. For either membrane system, the highest rate of [14C]cholesterol transfer was observed with the human and the rat HDL fraction. Since the mitochondria lack the receptors for HDL, one may conclude that the observed preferential transfer is not governed by a receptor-controlled interaction of HDL with the membrane. Under conditions where the pool of free cholesterol in the lipoprotein fractions was the same, HDL was a much more efficient acceptor of [14C]cholesterol from mitochondria than LDL or VLDL. Similarly, transfer of [14C]cholesterol proceeded at a higher rate to HDL than to sonicated egg phosphatidylcholine (PC) vesicles, even under conditions where there was a tenfold excess of the vesicle-PC pool over the HDL phospholipid pool. This preferred transfer of [14C]cholesterol to HDL cannot be explained by a random diffusion of monomer cholesterol molecules. Rather, it shows that HDL has a specific effect on this process in the sense that it most likely enhances the efflux of cholesterol from the membrane. Treatment of HDL with trypsin reduced the rate of [14C]cholesterol transfer by 40-50%, indicating that protein component(s) are involved. One of these components appears to be apoA-I, as this protein was shown to enhance the transfer of [14C]cholesterol from mitochondria to lipid vesicles.
Reverse cholesterol transport
High-density lipoprotein
Phospholipid transfer protein
Intermediate-density lipoprotein
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The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.
Reverse cholesterol transport
Intermediate-density lipoprotein
Low-density lipoprotein
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Citations (15)