Interaction between plasma lipoproteins and bilayer lipid membranes. The role of surface charge

Interactions of planar BLM with different thickness and surface charge were analysed theoretically. Drawing together of the membranes is accompanied with the appearance of intramembrane potential jumps which may cause destruction and breakdown of the membranes. The theory is extrapolated to the interaction between spherical lipoprotein particles and planar BLM. Experimentally calculated (by means of ESR) surface charges of lipoproteins of low density (LLD) (--0,3 . 10(-2) C/m2) and lipoproteins of high density (LHD) (--2 . 10(-2) C/m2) enabled calculation of the interaction energy between the particles and BLM as well as of the values of intramembrane potential jumps. The latter cause local reconstructions of the membranes in the contact region and fusion of the particles with them. The earlier obtained experimental data were proved by the finding that LHD adsorption as compared with LLD is impeded due to the existence of a high energetic barrier. These peculiarities of the particles manifested during their interactions with BLM seem to be one of the factors responsible for atherogenic function of LLD and antiatherogenic one of LHD.     

Surface charge of plasma lipoproteins from the data of potentiometric studies

The method of potentiometric titration is used to measure a surface very low, low- and high-density charge of the human blood plasma lipoproteins. Density of the surface charges is -0.5 X 10(-2); -0.2 X 10(-2); -3.4 X 10(-2) K/m2, respectively. The surface area of the lipoproteins determined by the method of radiation-free energy transfer between fluorescent probes amounts to 185.9; 241.7 and 426.8 m2 per 1 g of lipoprotein, respectively. Results of the experiments satisfactorily coincide with the data, obtained by other methods.     

Fluorescent probe study of the spatial structure of membranes and lipoproteins

A review is devoted to principles of studies in spatial structure of the model and biological membranes and lipoproteins on the basis of measuring radiationless energy transfer between fluorescent probes and from proteins to the probes. Recently the theory has been developed for energy transfer in membranes of various geometry and in lipoproteins of different size and structure. Special fluorescent probes are designed and made. The measurement procedure was tested in simulated systems and used to study a series of membranes as well as blood plasma lipoproteins of main classes. Everything above-mentioned resulted in obtaining data on the size of protein molecules in membranes and lipoproteins, proteins location relative to the lipid phase, on the surface area of the membranes (isolated and directly in a cell), association of protein molecules, state of near-protein lipid layer, membrane asymmetry, spreading of proteins on the lipoprotein surface, on the cholesterol effect on the lipid bilayer size etc.     

Characterization of the core and surface of human plasma lipoproteins. A study based on the use of five fluorophores.

The physical properties of the core and the surface of five classes of human plasma lipoproteins were investigated using five fluorescent probes. The location of the fluorescence probes in the lipoprotein assembly was determined using collisional quenching and resonance energy transfer. The fluorophores monitor different regions of the lipoproteins, as shown by fluorescence quenching. Diphenylhexatriene (DPH) and methyl trans-parinaric acid (MTPA), which are apolar molecules, are localized mainly in the lipoprotein core. Their distribution into the surface is dependent upon the volume ratio of the hydrophobic part of the envelope and the core. The polar fluorophores, trimethylaminodiphenylhexatriene (TMADPH), hydroxycoumarin (HC) and trans-parinaric acid (TPA) are anchored in the glycerol skeleton region of the surface monolayer with the fluorophore group of HC in the headgroup region of the phospholipids. We determined the temperature-dependent steady-state fluorescence anisotropy (r) of these fluorophores in the four major classes of lipoproteins: VLDL, LDL, HDL2, HDL3 and in abnormal HDL from abetalipoproteinemia patients (HDLab). The hydrophobic probes, DPH and MTPA, reported the r values in the lipoproteins in the following order: LDL greater than HDL2 greater than HDL3 much greater than VLDL. This order correlates with the triglyceride-to-cholesterol ester (TG/CE) ratio in the core of lipoproteins. The polar probes HC, TPA and TMADPH reported the r value in a different order: HDL2, HDL3 greater than or equal to LDL much greater than VLDL. This is compatible with the decreasing order of the protein to lipid ratio in the envelope of these lipoproteins. HDLab was investigated by three fluorescent probes: DPH, TMADPH and HC. The anisotropy of DPH in HDLab was larger than that of either HDL2 or HDL3 in normal donors, probably due to the smaller TG/CE ratio in HDLab. The lower r values reported by HC and TMADPH for HDLab are not fully understood and may be related to other factors such as acyl chains composition. The characterization of lipoproteins by fluorescence depolarization using probes of known location in the lipoprotein assembly is very sensitive and may be used to report deviation from the norm.     

Crystallization effects in serum high density lipoproteins

Peculiarities of aggregation in the samples of high density serum lipoproteins LHD2 and LHD3 obtained from healthy donors and patients with ishaemic heart disease were studied under isothermal exposition. It was found that lipoprotein samples from defective serum were characterized by chemical and structural inhomogeneity. It is reflected in the formation of spherolites of various types and of well-grown dendrite forms. Growth of stepped lens-shaped crystals of cholesterol takes place in the normal serum LHD3 preparations after long exposition. Crystal growth is thought to begin according to dislocational mechanism on linear defects existing in the liquid crystalline phase; then transformation to kinetically rough surface and to normal growth of the face occurs with the concentration increase.     

Increases in platelet and red cell counts,blood viscosity,and arterial pressure during mild surface cooling: factors in mortality from coronary and cerebral thrombosis in winter.

Six hours of mild surface cooling in moving air at 24 degrees C with little fall in core temperature (0.4 degree C) increased the packed cell volume by 7% and increased the platelet count and usually the mean platelet volume to produce a 15% increase in the fraction of plasma volume occupied by platelets. Little of these increases occurred in the first hour. Whole blood viscosity increased by 21%; plasma viscosity usually increased, and arterial pressure rose on average from 126/69 to 138/87 mm Hg. Plasma cholesterol concentration increased, in both high and low density lipoprotein fractions, but values of total lipoprotein and lipoprotein fractions were unchanged. The increases in platelets, red cells, and viscosity associated with normal thermoregulatory adjustments to mild surface cooling provide a probable explanation for rapid increases in coronary and cerebral thrombosis in cold weather. The raised arterial pressure and possibly cholesterol concentration may contribute to slower components of the increased thrombosis.     

Helix-helix association of a lipid-bound amphipathic alpha-helix derived from apolipoprotein C-II.

The interaction of a peptide derived from the sequence of apolipoprotein C-II (apoC-II) with a model lipid surface has been investigated by fluorescence spectroscopy. ApoC-II19-39, labeled at the N-terminus with 7-nitrobenz-2-oxa-1,3-diazole (NBD), bound to small unilamellar vesicles of phosphatidylcholine with a dissociation constant of 6 microM. The lipid-bound NBD-labeled peptide exhibited a red-edge excitation shift in its emission maximum and anisotropy, consistent with insertion of the probe into the motionally restricted, polar environment provided by the bilayer interface. The small Stokes shift of the NBD fluorophore permits electronic energy homotransfer between peptides on the lipid surface and results in depolarization of the NBD emission. At high surface densities of lipid-bound peptide, the anisotropy of the NBD probe was 33% lower than in corresponding samples in which electronic energy homotransfer was prevented by the addition of an unlabeled peptide. The efficiency of energy transfer between probes was not consistent with a random distribution of peptides on the lipid surface, indicating instead the self-association of lipid-bound apoC-II19-39. We propose that the role of this sequence in apoC-II is not only to mediate binding of protein to a lipid surface, but also to stabilize the lipoprotein complexes by associating with other amphipathic helices within apoC-II and with other apolipoproteins.     

Chromium picolinate and conjugated linoleic acid do not synergistically influence diet- and exercise-induced changes in body composition and health indexes in overweight women

This study assessed the effects of combined chromium picolinate (CP) and conjugated linoleic acid (CLA) supplementation on energy restriction and exercise-induced changes in body composition, glucose metabolism, lipid lipoprotein profile and blood pressure in overweight, premenopausal women. For 12 weeks, 35 women [age 36+/-1 years (mean+/-S.E.M.); BMI 28.0+/-0.5 kg/m2] were counseled to consume a 2092 kJ/day (500 kcal/day) energy deficit diet and performed 30 min of moderate-intensity walking or jogging 5 days/week. The women were randomly assigned to ingest either CP-CLA [400 mug chromium (Cr), 1.8 g CLA in 2.4 g tonalin oil, n=19] or placebo (<0.1 microg Cr, 2.4 g canola oil, n=16). Compared to baseline, urinary Cr excretion increased 22-fold, plasma CLA isomer 18:2 (c9,t11) content increased 79% and plasma CLA isomer 18:2 (t10,c12) became detectable in CP-CLA and were unchanged in Placebo. Over time, body weight decreased 3.5+/-0.5% (CP-CLA -2.6+/-0.5; placebo -2.5+/-0.5 kg) and fat mass decreased 8.9+/-1.3% (CP-CLA -2.7+/-0.5, placebo -2.4+/-0.5 kg), with no differences in responses between groups. Fasting blood hemoglobin A1c, plasma glucose and insulin, a homeostatic assessment of insulin resistance, serum total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol, triacylglycerol (TG), CHOL/HDL ratio, TG/HDL ratio and sitting systolic and diastolic blood pressures were not changed over time or influenced by CP-CLA. The use of a combined CP and CLA supplement for 3 months does not affect diet- and exercise-induced changes in weight and body composition or improve indexes of metabolic and cardiovascular health in young overweight women.     

Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.

125I-labeled and ferritin-labeled low density lipoprotein (LDL) were used as visual probes to study the surface distribution of LDL receptors and to examine the mechanism of the endocytosis of this lipoprotein in cultured human fibrobasts. Light microscopic autoradiograms of whole cells incubated with 125I-LDL at 4 degrees C showed that LDL receptors were widely but unevenly distributed over the cell surface. With the electron microscope, we determined that 60-70% of the ferritin-labeled LDL that bound to cells at 4 degrees C was localized over short coated segments of the plasma membrane that accounted for no more than 2% of the total surface area. To study the internalization process, cells were first allowed to bind ferritin-labeled LDL at 4 degrees C and were then warmed to 37 degrees C. Within 10 min, nearly all the surface-bound LDL-ferritin was incorporated into coated endocytic vesicles that were formed by the invagination and pinching-off of the coated membrane regions that contained the receptor-bound LDL. With increasing time at 37 degrees C, these coated vesicles were observed sequentially to migrate through the cytoplasm (1 min), to lose their cytoplasmic coat (2 min), and to fuse with either primary or secondary lysosomes (6 min). The current data indicate that the coated regions of plasma membrane are specialized structures of rapid turnover that function to carry receptor-bound LDL, and perhaps other receptor-bound molecules, into the cell.     

Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase

Lipoprotein lipase and the receptor-associated protein (RAP) bind to overlapping sites on the low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). We have investigated if lipoprotein lipase interacts with the RAP binding but structurally distinct receptor sortilin/neurotensin receptor-3. We show, by chemical cross-linking and surface plasmon resonance analysis, that soluble sortilin binds lipoprotein lipase with an affinity similar to that of LRP. The binding was inhibited by heparin and RAP and by the newly discovered sortilin ligand neurotensin. In 35S-labeled 3T3-L1 adipocytes treated with the cross-linker dithiobis(succinimidyl propionate), lipoprotein lipase-containing complexes were isolated by anti-sortilin antibodies. To elucidate function in cells, sortilin-negative Chinese hamster ovary cells were transfected with full-length sortilin and shown to express about 8% of the receptors on the cell surface. These cells degraded 125I-labeled lipoprotein lipase much faster than the wild-type cells. The degradation was inhibited by unlabeled lipoprotein lipase, indicating a saturable pathway, and by RAP and heparin. Moreover, inhibition by the weak base chloroquine suggested that degradation occurs in an acidic vesicle compartment. The results demonstrate that sortilin is a multifunctional receptor that binds lipoprotein lipase and, when expressed on the cell surface, mediates its endocytosis and degradation.     

Single-nucleotide polymorphism analysis using fluorescence resonance energy transfer between DNA-labeling fluorophore,fluorescein isothiocyanate,and DNA intercalator,POPO-3, on bacterial magnetic particles

To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*1/*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system.     

Effects of positive end-expiratory pressure on hepatic blood flow and performance

Positive end-expiratory pressure (PEEP) may impair extrapulmonary organ function. However, the effects of PEEP on the liver are unclear. We tested the hypothesis that at a constant cardiac output (CO), PEEP does not induce changes in hepatic blood flow (QL) and parenchymal performance. In splenectomized, close-chested canine preparations (group I, n = 6), QL was derived as hepatic outflow using electromagnetic flow probes (QLemf), and hepatic performance was defined by extraction and clearance of indocyanine green (ICG). In a noninvasive model (group II, n = 7), the effects of PEEP on hepatic performance alone were similarly analyzed. Measurements were taken during intermittent positive-pressure ventilation (IPPV1), after addition of 10 cmH2O PEEP to IPPV (PEEP1), during continued PEEP but after return of CO to IPPV1 levels by intravascular volume infusions (PEEP2), and after removal of both PEEP and excess blood volume (IPPV2). Phasic inspiratory decreases in QLemf present during positive-pressure ventilation were not increased during either PEEP1 or PEEP2. Mean QLemf decreased proportionately with CO during PEEP1 (P less than 0.05), but was restored to IPPV1 levels in a parallel fashion with CO during PEEP2. The ICG pharmacokinetic responses to PEEP were complex, with differential effects on extraction and clearance. Despite this, hepatic performance was not imparied in either group. we conclude that global QL reductions during PEEP are proportional to PEEP-induced decreases in CO and are preventable by returning CO to pre-PEEP levels by intravascular volume infusions. However, covarying changes in blood volume and hepatic outflow resistance may independently modulate hepatic function.     

Parinaroyl and pyrenyl phospholipids as probes for the lipid surface layer of human low density lipoproteins

A simple protocol employing lipid transfer proteins was developed to label human low density lipoprotein (LDL) in a controlled manner with parinaroyl and pyrenyl phosphatidylcholines. In order to study the lipid fluidity in the surface lipid layer of LDL, the temperature-dependence of both polarization (parinaroyl probes) and excimer to monomer (E/M) intensity ratio (pyrenyl probes) were analyzed. A series of pyrenyl phosphatidylcholines containing a pyrenyl fatty acid varying from 6 to 14 carbons in length at the sn-2 position were inserted into LDL to investigate the lateral distribution of different phosphatidylcholines in the lipoprotein surface at 37 degrees C. Both polarization and E/M vs. temperature plots displayed discontinuities in the region of 22-32 degrees C, which coincides with the melting of the neutral lipid core, indicating that the latter induces an ordered to more disordered phase transition in the surface lipid layer. Determination of the E/M intensity ratio as a function of pyrene lipid concentration in LDL showed a linear relationship for the pyrenyl hexanoate and octanoate species, whereas a slope discontinuity was observed for the lipids containing a longer pyrenyl chain. These data suggest that two lipid domains with distinct properties exist in the surface layer and secondly, pyrenyl lipids partition between these domains in a chainlength-dependent manner. This is consistent with measurement of the tryptophan to pyrene energy transfer efficiency vs. pyrenyl lipid concentration, which showed a biphasic relationship for the long-chain pyrenyl lipids. These measurements further indicate that two surface lipid domains correspond to the protein-lipid boundary and the bulk lipid phase, respectively. The fact that relatively small changes in chainlength have a marked influence on the partitioning of pyrenyl lipids between the boundary and the bulk phase suggests also that native phospholipid species may not be randomly distributed in the surface lipid layer of LDL.     

Estimation of surface area in very low density human serum lipoproteins

The surface area of very low density lipoproteins (VLDL) from the serum of 15 healthy donors and the surface area of artificial lipid particles have been estimated. The artificial particles were prepared as a mixture of egg phosphatidylcholine and triolein. Two fluorescent probes - energy donor and acceptor - were placed on the surface, and Forster's nonradiative energy transfer was measured; the transfer efficiency is a function of surface area. The fluorescent probe K-68 (4-[5-(phenyloxazolyl-2)-1-pentadecyl)pyridinium) was used as a donor, and DSP-12 (dimethylamino)styryl-N-dodecylpyridinium) was used as an acceptor. The specific surface area of the artificial lipid particles was estimated to be 0.585 +/- 0.015 nm2 per phosphatidylcholine molecule, which is 15% less than in lipid bilayers. The specific area of VLDL particles was 259 +/- 65 m2 per g of total VLDL. This value is close to the specific area of low density lipoproteins (LDL), and corresponds to the area of a spherical particle 10-12 nm in radius. However, VLDL are assumed to be much larger particles as compared with LDL. Therefore, the new data of the VLDL surface area raise a problem of revision of the existing VLDL models.     

The acute effects of low-intensity exercise on plasma lipids in endurance-trained and untrained young adults.

The acute effects of low-intensity exercise on plasma lipids were assessed in 22 healthy, normolipidaemic volunteers [mean age (SEM) 21.1 (0.2) years] of whom 11 were untrained and 11 endurance trained. Each subject walked for 2 h on a treadmill at a speed selected to elicit 30% [29.8 (3.9)%] of his or her maximal oxygen uptake. All subjects consumed a similar diet, i.e. 48% of energy from carbohydrate, for 2 days prior to the test. Pre-exercise, high-density lipoprotein (HDL) cholesterol concentration was higher in the trained group than in the untrained group [0.88 (0.06) mmol.l-1 vs 0.73 (0.09) mmol.l-1, P less than 0.05]. The walk elicited an increase in blood lactate concentration (P less than 0.01) but glucose homeostasis was well maintained by both groups. After 2 h of walking total cholesterol had increased by 13 (0.6)% (P less than 0.05). HDL cholesterol concentration increased by 17 (1.6)%, so that the ratio of total to HDL cholesterol was lower after the walk than pre-exercise (P less than 0.05). In the endurance-trained group HDL cholesterol concentration increased progressively, being 7.9 (2.4)% higher after 1 h and 19.7 (1.6)% higher after 2 h. A different response was evident in the untrained group where a rise after the 1st h [25.1 (2.3)%] was followed by a decrease towards pre-exercise values. These results show that one prolonged bout of low-intensity exercise modifies lipoprotein metabolism and hold out the interesting possibility that this response may differ in trained and untrained individuals.     

ApoA-I structure on discs and spheres. Variable helix registry and conformational states

Apolipoprotein A-I (apoA-I) readily forms discoidal high density lipoprotein (HDL) particles with phospholipids serving as an ideal transporter of plasma cholesterol. In the lipid-bound conformation, apoA-I activates the enzyme lecithin:cholesterol acyltransferase stimulating the formation of cholesterol esters from free cholesterol. As esterification proceeds cholesterol esters accumulate within the hydrophobic core of the discoidal phospholipid bilayer transforming it into a spherical HDL particle. To investigate the change in apoA-I conformation as it adapts to a spherical surface, fluorescence resonance energy transfer studies were performed. Discoidal rHDL particles containing two lipid-bound apoA-I molecules were prepared with acceptor and donor fluorescent probes attached to cysteine residues located at specific positions. Fluorescence quenching was measured for probe combinations located within repeats 5 and 5 (residue 132), repeats 5 and 6 (residues 132 and 154), and repeats 6 and 6 (residue 154). Results from these experiments indicated that each of the 2 molecules of discoidal bound apoA-I exists in multiple conformations and support the concept of a "variable registry" rather than a "fixed helix-helix registry." Additionally, discoidal rHDL were transformed in vitro to core-containing particles by incubation with lecithin:cholesterol acyltransferase. Compositional analysis showed that core-containing particles contained 11% less phospholipid and 633% more cholesterol ester and a total of 3 apoA-I molecules per particle. Spherical particles showed a lowering of acceptor to donor probe quenching when compared with starting rHDL. Therefore, we conclude that as lipid-bound apoA-I adjusts from a discoidal to a spherical surface its intermolecular interactions are significantly reduced presumably to cover the increased surface area of the particle.     

Mapping QTLs for heading synchrony in a doubled haploid population of rice in two environments

Simultaneous heading of plants within the same rice variety, also refer to heading synchrony, is an important factor that affects simultaneous ripening of the variety. Understanding of the genetic basis of heading synchrony may contribute to molecular breeding of rice with simultaneous heading and ripening. In the present study, a doubled haploid （DH） population, derived from a cross between Chunjiang 06 and TN1 was used to analyze quantitative trait locus （QTL） for heading synchrony related traits, i.e., early heading date （EHD）, late heading date （LHD）, heading asynchrony （HAS）, and tiller number （PN）. A total of 19 QTLs for four traits distributed on nine chromosomes were detected in two environments. One QTL, qHAS-8 for HAS, explained 27.7% of the phenotypic variation, co-located with the QTLs for EHD and LHD, but it was only significant under long-day conditions in Hangzhou, China. The other three QTLs, qHAS-6, qHAS-9, and qHAS-10, were identified under short-day conditions in Hainan, China, each of which explained about 11% of the phenotypic variation. Two of them, qHAS-6 and qHAS-9, were co-located with the QTLs for EHD and LHD. Two QTLs, qPN-4 and qPN-5 for PN, were detected in Hangzhou, and qPN-5 was also detected in Hainan. However, none of them was co-located with QTLs for EHD, LHD, and HAS, suggesting that PN and HAS were controlled by different genetic factors. The results of this study can be useful in marker assisted breeding for improvement of heading synchrony.     

下丘脑Orexin 对肥胖大鼠能量代谢的影响

目的:探讨下丘脑注射OXR-1选择性受体拮抗剂ACT-335827对肥胖大鼠代谢的效果。方法:通过高脂饮食建立肥胖大鼠模型,采用CODA 8通道高通量非侵入性血压系统(EMKA)测量血压;所有脂类都使用商品酶试剂盒和TOSHIBA-40FR全自动分析仪测量;空腹血糖采用葡萄糖氧化酶法;空腹胰岛素采用放射免疫法测定。肥胖大鼠出现代谢紊乱后,给予ACT-335827处理,检测大鼠体重、血压、脂肪、甘油三酯、总胆固醇、高密度脂蛋白、低密度脂蛋白、游离脂肪酸(NEFA)、瘦素、空腹血糖及空腹胰岛素等的变化。结果:与普通饮食组相比,经过10周高脂饮食,高脂饮食组大鼠体重显著升高(P0.05),给予ACT-335827处理后,普通大鼠的体重、血压、脂肪含量、脂代谢等均无明显变化;与高脂饮食和高脂饮食加生理盐水处理组大鼠比较,高脂饮食加ACT-335827处理组肥胖大鼠的体重显著下降(P0.05),腹部和附睾脂肪含量下降(P0.05),低密度脂蛋白、甘油三酯、总胆固醇、瘦素水平下降(P0.05),空腹血糖及空腹胰岛素也显著降低(P0.05),但血压、肠系膜脂肪和肩胛棕色脂肪、高密度脂蛋白和NEFA无明显变化(P0.05)。结论:ACT-335827对肥胖大鼠的代谢紊乱具有改善作用,对肥胖大鼠有一定的减肥作用。     

Sphingomyelinase activity associated with human plasma low density lipoprotein

Isolated human plasma low density lipoprotein (LDL) was observed to possess sphingomyelinase activity. Accordingly, the formation of ceramide was catalyzed by LDL at 37 degrees C using tertiary liposomes composed of sphingomyelin (mole fraction (x) = 0.2), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (x = 0.7), 1, 2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (x = 0.1), and either the fluorescent sphingomyelin analog Bodipy-sphingomyelin or [(14)C]sphingomyelin as substrates. However, this activity was not present in either very low density lipoprotein or the high density lipoprotein subfractions HDL(2) and HDL(3). Oxidation of LDL abrogated its sphingomyelinase activity. Aggregation of the liposomes upon incubation with LDL was evident from the light scattering measurements. Microinjection of LDL to the surface of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-d-sphingomyelin (C16:0-sphingomyelin), and Bodipy-sphingomyelin as a fluorescent tracer (0.75:- 0.20:0.05, respectively) revealed the induction of vectorial budding of vesicles, resembling endocytosis.     

In vivo evidence of a role for hepatic lipase in human apoB-containing lipoprotein metabolism, independent of its lipolytic activity

Hepatic lipase (HL) is a key player in lipoprotein metabolism by modulating, through its lipolytic activity, the triglyceride (TG) and phospholipid content of apolipoprotein B (apoB)-containing lipoproteins and of high density lipoproteins (HDL), thereby affecting their size and density. A new and separate role has been suggested for HL in cellular lipoprotein metabolism, in which it serves as a ligand promoting cellular uptake of apoB-containing remnant lipoproteins and HDL. We tested the hypothesis that HL has both a lipolytic and a nonlipolytic role in human lipoprotein metabolism, by measuring lipid plasma concentrations, lipoprotein density distribution by density gradient ultracentrifugation, and lipoprotein composition, in three subjects with HL deficiency: two of the patients (S-1 and S-3) were characterized as having neither plasma HL activity nor detectable HL protein; the third subject (S-2) had no plasma HL activity but a detectable amount (35.5 ng/ml) of HL protein. All HL-deficient subjects showed a severalfold increase in lipoprotein TG content across the lipoprotein density spectrum [very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and HDL] as compared with control subjects. They also had remarkably more buoyant LDL particles (LDL-R(f) = 0.342;-0.394) as compared with the control subjects (LDL-R(f) = 0.303). Subjects S-1 and S-3 (no HL activity or protein) presented with a distinct increase in cholesterol and apoB levels in the IDL and VLDL density range as compared with patient S-2, with detectable HL protein, and the control subjects.This study provides evidence in humans that HL indeed plays an important role in lipoprotein metabolism independent of its enzymatic activity: in particular, inactive HL protein appears to affect VLDL and IDL particle concentration, whereas HL enzymatic activity seems to influence VLDL-, IDL-, LDL-, and HDL-TG content and their physical properties.