Packing of cholesterol molecules in human low-density lipoprotein

High-resolution, proton-decoupled 13C nuclear magnetic resonance spectra (90.55 MHz) of human low-density lipoprotein (LDL) have been employed to investigate the physical state of unesterified cholesterol molecules in this particle. Approximately half of the cholesterol molecules in LDL were replaced with [4-13C]cholesterol by exchange from Celite. About two-thirds of the cholesterol molecules contribute to a resonance at delta 41.8 from the C-4 atom. This signal is assigned to cholesterol molecules located at the surface of the LDL particle in a mixed monolayer with phospholipid molecules; the spin-lattice relaxation of the C-4 nucleus of such cholesterol molecules is enhanced by the presence of Mn2+ ions in the aqueous phase. The remaining one-third of the cholesterol molecules are apparently neither associated with phospholipid nor exposed to the aqueous phase; these cholesterol molecules are presumed to be located in the core of the particle. Cholesterol molecules in the two microenvironments are in slow exchange on the NMR time scale but in fast exchange on a biological time scale, so that the cholesterol molecules in LDL behave physiologically as one pool. There is a loss of about 20% of the intensity of the N(CH3)3 resonance from phosphatidylcholine and sphingomyelin molecules in the LDL spectrum; this is attributed to the presence of apolipoprotein B in the surface of LDL particles, which may immobilize some of the phospholipid polar groups. Spin-lattice relaxation time measurements suggest that the fast axial motions of cholesterol molecules in the surface of LDL are the same as in high-density lipoprotein (HDL).(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein oxidative modification induces procoagulant profiles in endogenous hypertriglyceridemia

This study was to investigate whether oxidatively modified lipoproteins were associated with changes of pro- and anticoagulant profiles in hypertriglyceridemic subjects. Plasma VLDL, LDL, and HDL were isolated with the one-step density gradient ultracentrifugation method. The oxidation of the lipoproteins was identified. Prothrombin time (PT) and activated partial thrombplastin time (APTT), tissue plasminogen activator and plasminogen activator inhibitor-1, and platelet aggregation rate were determined with a reaction system consisting of mixed fresh normal plasma, in endogenous hypertriglyceridemic (HTG) patients, in in vitro modified lipoproteins from a normolipidemic donor, and in experimental rats. The results indicated that oxVLDL, oxLDL, and oxHDL occurred in the plasma of HTG patients. Compared with the control group, PT and APTT, incubated with plasma VLDL, LDL, or HDL from HTG patients, respectively, were significantly reduced, while platelet maximal aggregation rates were significantly higher (P < 0.05-0.01). Similar procoagulant profiles were observed in in vitro modified lipoprotein components and in rats with intrinsic hypertriglyceridemia as well. These results support our previous finding that LDL, VLDL, and HDL were all oxidatively modified in vivo in the subjects with HTG, and suggest that procoagulation state may result from the abnormal plasma lipoprotein oxidative modification in vivo.     

The low-density lipoprotein pathway of cultured Leydig tumor cells. Utilization of low-density lipoprotein-derived cholesterol for steroidogenesis

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.     

Physical training and plasma intermediate density lipoprotein cholesterol levels in men with hypertriglyceridemia

Plasma lipoprotein lipid levels including IDL cholesterol concentration, physical fitness parameters, and anthropometric data were measured in 10 men with primary hypertriglyceridemia during a 12 week program of moderate physical training. Reduced pulse rate at submaximal workload and increased work performed on the treadmill indicated that the men had increased their physical fitness during the training period. Body weight (-4.9%) and total skinfold thickness (-15.6%) fell during the training program. A characteristic fall (-23%) in plasma triglyceride levels was seen over the first 8 weeks training. Plasma IDL cholesterol concentration fell (-18%) after 2 weeks training but then rose after 8 weeks to levels which tended to be higher than baseline values. Our data suggest that regular physical exercise is more likely to raise levels of the "proatherogenic" IDL than to lower them in hypertriglyceridemic patients.     

Independent associations between low-density lipoprotein cholesterol and cancer among patients with type 2 diabetes mellitus

Background

The risk association between low-density lipoprotein (LDL) cholesterol and cancer remains controversial and largely unexplored for people not receiving statin therapy.

Methods

We examined the risk association between LDL cholesterol and cancer among patients with type 2 diabetes mellitus who were free of cancer at enrolment and whose statin use was known. We considered a variety of nonlinear relationships in our analysis.

Results

During a median follow-up period of 4.90 years, cancer developed in 270 (4.4%) of 6107 patients. Among the 3800 patients who did not receive statin therapy, the risk association between LDL cholesterol and cancer was represented by a V-shaped curve. Compared with patients whose LDL cholesterol was at least 2.80 mmol/L but less than 3.80 mmol/L, the risk of cancer, death from any cause or the composite outcome of cancer or death was greater among those with an LDL cholesterol level of less than 2.80 mmol/L (hazard ratio for cancer 1.74, 95% confidence interval [CI] 1.20–2.52) and those with an LDL cholesterol level of 3.80 mmol/L or greater (hazard ratio for cancer 1.87, 95% CI 1.29–2.71). Using 3.8 mmol/L as a reference point, we found that the hazard ratio for cancer for every millimole per litre absolute change in LDL cholesterol was 1.54 (95% CI 1.19–1.99) among patients not using statins; the hazard ratio was reduced to 1.24 (1.01–1.53) for the entire sample (statin users and those not using statins). These associations persisted after adjustment for covariates and exclusion of patients with less than 2.5 years of follow-up.

Interpretation

Among patients with type 2 diabetes, the association between LDL cholesterol and cancer was V-shaped, whereby both low and high levels of LDL cholesterol were associated with elevated risk of cancer.Emerging data suggest an association between diabetes mellitus and an increased risk of cancer,¹ including breast cancer in women;² colorectal,³ pancreatic^4,5 and liver⁶ cancer in both men and women; and prostate cancer in men.⁷ Several prospective analyses, including the US National Health and Nutrition Examination Survey,⁸ have demonstrated an inverse relation between serum total cholesterol and cancer incidence and mortality in the general population, although few studies have investigated this relation among patients with type 2 diabetes mellitus. In 2004, the US National Cholesterol Education Program Adult Treatment Panel III recommended treatment targets for low-density lipoprotein (LDL) cholesterol of less than 1.81 mmol/L (less than 70 mg/dL) for patients with very high risk of coronary artery disease and less than 2.59 mmol/L (less than 100 mg/dL) for patients with high risk of coronary artery disease;⁹ these targets were intended for both diabetic and nondiabetic patients.These recommendations remain controversial. Although one recent analysis of a large cohort of patients treated with statins showed a greater risk of cancer with achievement of low LDL cholesterol,¹⁰ a more recent study reported otherwise.¹¹ Furthermore, the independent associations between LDL cholesterol level and cancer in both the general population and in patients with type 2 diabetes have not been explored. We conducted a hypothesis-generating study to explore the possible independent association between LDL cholesterol and cancer risk in Chinese patients with type 2 diabetes mellitus.     

Lecithin: cholesterol acyltransferase is insufficient to prevent oxidative modification of low-density lipoprotein.

We tried to confirm the antioxidative capability of lecithin:cholesterol acyltransferase (LCAT) reported by Vohl et al. [Biochemistry (1999) 38, 5976-5981]. The enzyme solution protected LDL against oxidation. However, this protection was not due to LCAT enzyme, but to some unknown low-molecular-weight substance(s) in the solution; LCAT itself exerted little protective effect against LDL oxidation.     

Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase

The metabolism of cholesterol derived from [3H]cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large alpha-migrating HDL (HDL2) and was then transferred to small alpha-HDL (HDL3) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating in HDL2. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL2 as a relatively unreactive sink for LCAT-derived cholesteryl esters.     

Homocysteine in relation to C-reactive protein and low-density lipoprotein cholesterol in assessment of cardiovascular risk.

Coronary vascular disease (CVD) is a chronic, multifactorial disease that occurs often in individuals without known risk factors. We investigated the predictive value of homocysteine (Hcy) in relation to C-reactive protein (CRP) and low-density lipoprotein (LDL)-cholesterol in patients with confirmed coronary disease. The study included 87 German and 92 Syrian patients in addition to 87 German and 64 Syrian control individuals. Patients and controls were of comparable age, lifestyles and cultural background. Patients of both ethnic groups had significantly higher concentrations of Hcy and C-reactive protein compared to the controls. The lipids were higher only in Syrian patients compared to the controls. Elevated concentrations of Hcy or that of CRP (>75th percentiles) were associated with increased probability for CVD. In both population groups, the risk increased markedly in subjects who had elevated concentrations of Hcy and CRP or those who had elevated concentrations of Hcy and LDL-cholesterol. The results emphasize that detemination of Hcy may improve the predictive value of C-reactive protein and the LDL-cholesterol. Measurements of these markers are especially important for identification of patients at high risk for CVD.     

Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.     

Measurement of receptor-independent metabolism of low-density lipoprotein. An application of glycosylated low-density lipoprotein

Incubation of human low-density lipoprotein (LDL) with glucose results in a nonenzymatic formation of a Schiff base between the monosaccharide and lysyl residues of apolipoprotein B. Increasing the percentage of lysyl residues of apolipoprotein B modified by glycosylation decreases the fractional catabolic rate of the glycosylated LDL, and decreases the metabolism of the glycosylated LDL by human skin fibroblasts. The glycosylated LDL, containing 20-40% of total lysyl residues of apoprotein B modified, was metabolized at a slow rate by both human skin fibroblasts and mouse peritoneal macrophages. These results led to the suggestion that glycosylated LDL is primarily catabolized via a receptor-independent process. Assuming LDL catabolism occurs via receptor-dependent and receptor-independent processes, the ratio of (fractional catabolic rate of glycosylated LDL)/(fractional catabolic rate of native LDL) should be an estimate of the percentage of LDL catabolism via the receptor-independent process. From the fractional catabolic rates of glucose-LDL (20-40% of lysyl residues modified) and galactose-LDL (30-60% of lysyl residues modified) 41% and 30% respectively, of LDL catabolism occurred by a receptor-independent process.     

Effect of antioxidant protection by p-coumaric acid on low-density lipoprotein cholesterol oxidation

Mechanisms inwhich p-coumaric acid (CA) acts as an antioxidant are notwell understood. This study investigated whether CA can act as a directscavenger of reactive oxygen species (ROS) and whether it minimizes theoxidation of low-density lipoprotein (LDL). Rats were administered CAin drinking water at low or high doses for 10, 21, and 30 days (uptakeswere 29 and 317 mg/day, respectively). Blood levels of8-epiprostaglandin F₂ were monitored as a marker of LDLoxidation. Oral administration of CA (317 mg/day) for 30 dayssignificantly inhibited LDL oxidation. CA also reduced LDL cholesterollevels in serum but had no effect on levels of high-density lipoproteincholesterol. In vitro studies that used electron spin resonance incombination with spin trapping techniques were used to determine theability of CA to scavenge ROS and alter LDL oxidation. CA effectivelyscavenged ·OH in a dose-dependent manner. IC₅₀ andmaximum velocity for CA scavenging of ·OH were 4.72 µM and 1.2 µM/s, respectively, with a rate constant of 1.8 × 10¹¹ M¹ · s¹. Ourstudies suggest that the antioxidant properties of CA may involve thedirect scavenging of ROS such as ·OH.

     

The effects of low-density lipoprotein and cholesterol on acyl-coenzyme A: cholesterol acyltransferase activity in membranes from cultured human fibroblasts.

Membranes prepared from cultured fibroblasts were assayed for acyl-coenzyme A: cholesterol acyltransferase (ACAT) by a method that relied exclusively on the cholesterol already present on the membranes as the sterol substrate. Changes in membrane ACAT activity during incubation of fibroblasts under a variety of conditions were similar to the changes in the rate of incorporation of oleic acid into cholesteryl esters by the intact cells. The addition of low-density lipoprotein (LDL) to fibroblasts pre-incubated with lipoprotein-deficient serum led to a transient increase in membrane ACAT activity, which reached its peak after 7h and was related to the receptor-mediated uptake and degradation of the lipoprotein by the cells. However, after incubation of the membranes with a cholesterol-rich donor lipoprotein, which resulted in an equilibration of cholesterol between membranes and donor, each preparation exhibited the same activity. In contrast with these effects of LDL, incubation of the cells with non-esterified cholesterol produced a prolonged increase in ACAT activity and an increase in the activity observed after equilibration. Furthermore, ACAT activity in cells grown with linoleic acid was higher, both before and after the addition of LDL, than that of cells grown in normal medium or with palmitate. The increase in activity produced by LDL was also greater, reflecting the greater rate of degradation of LDL by the cells, and was associated with an increase in the activity observed after equilibration with donor. The results suggest that although fibroblasts can increase the amount of active enzyme on their membranes to accommodate an exceptionally high or prolonged supply of cholesterol, under normal circumstances the increase in membrane ACAT activity produced by LDL can be explained entirely by an increase in the amount of cholesterol in the substrate pool.     

HMG-CoA reductase inhibitor decreases small dense low-density lipoprotein and remnant-like particle cholesterol in patients with type-2 diabetes

Our studies have focused on the effect of injection of L-NAME and sodium nitroprussiate (SNP) on the salivary secretion, arterial blood pressure, sodium excretion and urinary volume induced by pilocarpine which was injected into the medial septal area (MSA). Rats were anesthetized with urethane (1.25 g/kg b. wt.) and a stainless steel cannula was implanted into their MSA. The amount of saliva secretion was studied over a five-minute period after injection of pilocarpine into MSA. Injection of pilocarpine (10, 20, 40, 80, 160 microg/microl) into MSA produced a dose-dependent increase in salivary secretion. L-NG-nitro arginine methyl-esther (L-NAME) (40 microg/microl), a nitric oxide (NO) synthase inhibitor, was injected into MSA prior to the injection of pilocarpine into MSA, producing an increase in salivary secretion due to the effect of pilocarpine. Sodium nitroprussiate (SNP) (30 microg/microl) was injected into MSA prior to the injection of pilocarpine into MSA attenuating the increase in salivary secretion induced by pilocarpine. Medial arterial pressure (MAP) increase after injections of pilocarpine into the MSA. L-NAME injected into the MSA prior to injection of pilocarpine into MSA increased the MAP. SNP injected into the MSA prior to pilocarpine attenuated the effect of pilocarpine on MAP. Pilocarpine (40 ug/ul) injected into the MAS induced an increase in sodium and urinary excretion. L-NAME injected prior to pilocarpine into the MSA increased the urinary sodium excretion and urinary volume induced by pilocarpine. SNP injected prior to pilocarpine into the MSA decreased the sodium excretion and urinary volume induced by pilocarpine. All these roles of pilocarpine depend on the release of nitric oxide into the MSA. We may also conclude that the MSA is involved with the cholinergic excitatory mechanism that induce salivary secretion, increase in MAP and increase in sodium excretion and urinary volume.     

Electronegative low-density lipoprotein

PURPOSE OF REVIEW: The occurrence in blood of an electronegatively charged LDL was described in 1988. During the 1990s reports studying electronegative LDL (LDL(-)) were scant and its atherogenic role controversial. Nevertheless, recent reports have provided new evidence on a putative atherogenic role of LDL(-). This review focuses on and discusses these new findings. RECENT FINDINGS: In recent years, LDL(-) has been found to be involved in several atherogenic features through its action on cultured endothelial cells. LDL(-) induces the production of chemokines, such as IL-8 and monocyte chemotactic protein 1, and increases tumor necrosis factor-alpha-induced production of vascular cell adhesion molecule 1, with these molecules being involved in early phases of leukocyte recruitment. LDL(-) from familial hypercholesterolemic patients also decreases DNA synthesis and intracellular fibroblast growth factor 2 production, which may contribute to impaired angiogenesis and increased apoptosis. In addition, the preferential association of platelet-activating factor acetylhydrolase with LDL(-) has been reported, suggesting a proinflammatory role of this enzyme in LDL(-). SUMMARY: Recent findings suggest that LDL(-) could contribute to atherogenesis via several mechanisms, including proinflammatory, proapoptotic and anti-angiogenesis properties. Further studies are required to define the role of LDL(-) in atherogenesis more precisely and to clarify mechanisms involved in endothelial cell activation.     

Incubation of acetylated low-density lipoprotein with cholesterol-rich dispersions enhances cholesterol uptake by macrophages

Incubation of J774 macrophages with mixtures of acetylated low-density lipoprotein (acLDL) and free cholesterol-rich phospholipid dispersions increases cellular cholesterol deposition 2-4-fold over that achieved with either acLDL or dispersions alone. Both free and esterified cholesterol accumulate in cells incubated with the mixture of acLDL and dispersions. A similar result is observed when acLDL is replaced by malondialdehyde-LDL. The enhanced deposition of cholesterol is not unique to J774 macrophages, as P388D1 macrophages also accumulate more cholesterol when incubated with the mixture of acLDL and dispersions than either particle alone. A preincubation of the particles for at least 6 h prior to incubation with cells is required in order to observe maximal cholesterol delivery. Both dispersion free cholesterol and phospholipid accumulate in J774 cells, suggesting that a complex is formed between acLDL and dispersions which results in a cholesterol-rich acLDL/dispersion particle. Partial purification of the acLDL-dispersion complex revealed increases in the size distribution of the particles compared to acLDL and increases in free cholesterol and phospholipid contents. Cholesterol uptake from the mixture of acLDL and dispersions was saturable and the enhanced cellular uptake of both cholesterol and phospholipid from the complex could be abolished by inhibitors of the scavenger receptor pathway. In addition to the receptor-mediated uptake of cholesterol from the acLDL-dispersion complex, it was observed that approx. 30% of the total cholesterol uptake from the complex was via non-specific components, including surface transfer.     

Lecithin:cholesterol acyltransferase reduces the adverse effects of oxidized low-density lipoprotein while incurring damage itself.

In this study, we tried to determine (a) whether there is any permanent effect of oxLDL on the LCAT molecule and its activator lipoprotein apoA-I, and (b) the fate of oxLDL after its exposure to LCAT and apoA-I. Purified LCAT and LDL/oxLDL was incubated at 37 degrees C for 1 h, and separated by gel-permeation chromatography. Its activity was significantly less (30% less) after separating from oxLDL compared to that of LDL. Cofactor activity of isolated apoA-I (incubated with LDL/oxLDL) was found affected by oxLDL. These results indicate modification of the LCAT and apoA-I molecule. But LCAT was found more affected compared to apoA-I in terms of LCAT activity. We are also reporting a novel function of LCAT. It was found to reduce the adverse effects of oxLDL, for example, ability to affect the LCAT activity and TBARS value. This ability of LCAT to repair oxLDL was dose-dependent. But there was no change in its REM values or fluorescence intensity.