Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse.

This study examines the question of whether apolipoprotein E (apoE) alters steady-state concentrations of plasma cholesterol carried in low density lipoproteins (LDL-C) by acting as a competitive inhibitor of hepatic LDL uptake or by altering the rate of net cholesterol delivery from the intestinal lumen to the liver. To differentiate between these two possibilities, rates of cholesterol absorption and synthesis and the kinetics of hepatic LDL-C transport were measured in vivo in mice with either normal (apoE+/+) or zero (apoE-/-) levels of circulating apoE. Rates of cholesterol absorption were essentially identical in both genotypes and equaled approximately 44% of the daily dietary load of cholesterol. This finding was consistent with the further observation that the rates of cholesterol synthesis in the liver (approximately 2,000 nmol/h) and extrahepatic tissues (approximately 3,000 nmol/h) were also essentially identical in the two groups of mice. However, the apparent Michaelis constant for receptor-dependent hepatic LDL-C uptake was markedly lower in the apoE-/- mice (44 +/- 4 mg/dl) than in the apoE+/+ animals (329 +/- 77 mg/dl) even though the maximal transport velocity for this uptake process was essentially the same (approximately 400 micrograms/h per g) in the two groups of mice. These studies, therefore, demonstrate that apoE-containing lipoproteins can act as potent competitive inhibitors of hepatic LDL-C transport and so can significantly increase steady-state plasma LDL-C levels. This apolipoprotein plays no role, however, in the regulation of cholesterol absorption, sterol biosynthesis, or hepatic LDL receptor number, at least in the mouse.

Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line.

The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.

The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.

We have previously reported the partial purification of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that binds oxidatively modified low density lipoprotein (OxLDL) and phosphatidylserine-rich liposomes. We have now identified that protein as macrosialin, a previously cloned macrophage-restricted membrane protein in the lysosomal-associated membrane protein family (mouse homologue of human CD68). Early in the course of purification of the 94- to 97-kDa protein, a new OxLDL-binding band at 190-200 kDa appeared and copurified with the 94- to 97-kDa protein. The HPLC pattern of tryptic peptides from this higher molecular mass ligand-binding band closely matched that derived from the 94- to 97-kDa band. Specifically, the same three macrosialin-derived tryptic peptides (9, 9, and 15 residues) were present in the purified 94- to 97-kDa band and in the 190- to 200-kDa band and antisera raised against peptide sequences in macrosialin recognized both bands. An antiserum against macrosialin precipitated most of the 94- to 97-kDa OxLDL-binding material. We conclude that the binding of OxLDL to mouse macrophage membranes is in part attributable to macrosialin. Our previous studies show that OxLDL competes with oxidized red blood cells and with apoptotic thymocytes for binding to mouse peritoneal macrophages. Whether macrosialin plays a role in recognition of OxLDL and oxidatively damaged cells by intact macrophages remains uncertain.

Mutant oocytic low density lipoprotein receptor gene family member causes atherosclerosis and female sterility.

The so-called very low density lipoprotein receptors (VLDLRs) are related to the LDLR gene family. So far, naturally occurring mutations have only been described for the prototype LDLR; in humans, they cause familial hypercholesterolemia. Here we describe a naturally occurring mutation in a VLDLR that causes a dramatic abnormal phenotype. Hens of the mutant restricted-ovulator chicken strain carry a single mutation, lack functional oocyte receptors, are sterile, and display severe hyperlipidemia with associated premature atherosclerosis. The mutation converts a cysteine residue into a serine, resulting in an unpaired cysteine and greatly reduced expression of the mutant avian VLDLR on the oocyte surface. Extraoocytic cells in the mutant produce higher than normal amounts of a differentially spliced form of the receptor that is characteristic for somatic cells but absent from germ cells.

The roles of coenzyme Q10 and vitamin E on the peroxidation of human low density lipoprotein subfractions.

The aim of our study was to investigate the relationships between the levels of coenzyme Q10 (CoQ10) and vitamin E and the levels of hydroperoxide in three subfractions of low density lipoproteins (LDL) that were isolated from healthy donors. LDL3, the densest of the three subfractions, has shown statistically significant lower levels of CoQ10 and vitamin E, which were associated with higher hydroperoxide levels when compared with the lighter counterparts. After CoQ10 supplementation, all three LDL subfractions had significantly increased CoQ10 levels. In particular, LDL3 showed the highest CoQ10 increase when compared with LDL1 and LDL2 and was associated with a significant decrease in hydroperoxide level. These results support the hypothesis that the CoQ10 endowment in subfractions of LDL affects their oxidizability, and they have important implications for the treatment of disease.

Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

The role of heritable, population-wide cell damage in neoplastic development was studied in the 28 L subline of NIH 3T3 cells. These cells differ from the 17(3c) subline used previously for such studies in their lower frequency of "spontaneous" transformation at high population density and their greater capacity to produce large, dense transformed foci. Three cultures of the 28 L subline of NIH 3T3 cells were held under the constraint of confluence for 5 wk (5 wk 1 degree assay) and then assayed twice in succession (2 degrees and 3 degrees assays) for transformed foci and saturation density. After the 2 degrees assay, the cells were also passaged at low density to determine their exponential growth rates and cloned to determine the size and morphological features of the colonies. Concurrent measurements were made in each case with control cells that had been kept only in frequent low-density passages and cells that had been kept at confluence for only 2 wk (2 wk 1 degree). Two of the three cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures produced light transformed foci, and the third produced dense foci. The light focus-forming cultures grew to twice the control saturation density in their 2 degrees assay and 6-8 times the control density in the 3 degrees assay; saturation densities for the dense focus formers were about 10 times the control values in both assays. All three of the cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures multiplied at lower rates than controls at low densities, but the dense focus formers multiplied faster than the light focus formers. The reduced rates of multiplication of the light focus formers persisted for > 50 generations of exponential multiplication at low densities. Isolated colonies formed from single cells of the light focus formers were of a lower population density than controls; colonies formed by the dense focus formers were slightly denser than the controls but occupied only half the area. A much higher proportion of the colonies from the 5 wk 1 degree cultures than the controls consisted of giant cells or mixtures of giant and normal-appearing cells. The results reinforce the previous conclusion that the early increases in saturation density and light focus formation are associated with, and perhaps caused by, heritable, population-wide damage to cells that is essentially epigenetic in nature. The more advanced transformation characterized by large increases in saturation density and dense focus formation could have originated from rare genetic changes, such as chromosome rearrangements, known to occur at an elevated frequency in cells destabilized by antecedent cellular damage.

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter.

Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance. The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs). Interestingly, both are essential for normal sterolmediated regulation of the promoter. The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process. SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA. In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation. The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter. Additionally, the C domain is also crucial. This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators. We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1. Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis.

Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo.

The 39-kDa receptor-associated protein (RAP) associates with the multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) and thereby prevents the binding of all known ligands, including alpha 2-macroglobulin and chylomicron remnants. RAP is predominantly localized in the endoplasmic reticulum, raising the possibility that it functions as a chaperone or escort protein in the biosynthesis or intracellular transport of LRP. Here we have used gene targeting to show that RAP promotes the expression of functional LRP in vivo. The amount of mature, processed LRP is reduced in liver and brain of RAP-deficient mice. As a result, hepatic clearance of alpha 2-macroglobulin is impaired and remnant lipoproteins accumulate in the plasma of RAP-deficient mice that also lack functional LDL receptors. These results are consistent with the hypothesis that RAP stabilizes LRP within the secretory pathway. They also suggest a further mechanism by which the activity of an endocytic receptor may be modulated in vivo.

Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.

Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP.

Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.

The influence of thermally induced changes in the lipid core structure on the oxidative resistance of discrete, homogeneous low density lipoprotein (LDL) subspecies (d, 1.0297-1.0327 and 1.0327-1.0358 g/ml) has been evaluated. The thermotropic transition of the LDL lipid core at temperatures between 15 degrees C and 37 degrees C, determined by differential scanning calorimetry, exerted significant effects on the kinetics of copper-mediated LDL oxidation expressed in terms of intrinsic antioxidant efficiency (lag time) and diene production rate. Thus, the temperature coefficients of oxidative resistance and maximum oxidation rate showed break points at the core transition temperature. Temperature-induced changes in copper binding were excluded as the molecular basis of such effects, as the saturation of LDL with copper was identical below and above the core transition. At temperatures below the transition, the elevation in lag time indicated a greater resistance to oxidation, reflecting a higher degree of antioxidant protection. This effect can be explained by higher motional constraints and local antioxidant concentrations, the latter resulting from the freezing out of antioxidants from crystalline domains of cholesteryl esters and triglycerides. Below the transition temperature, the conjugated diene production rate was decreased, a finding that correlated positively with the average size of the cooperative units of neutral lipids estimated from the calorimetric transition width. The reduced accessibility and structural hindrance in the cluster organization of the core lipids therefore inhibits peroxidation. Our findings provide evidence for a distinct effect of the dynamic state of the core lipids on the oxidative susceptibility of LDL and are therefore relevant to the atherogenicity of these cholesterol-rich particles.

Rapid Communication: Cholesterol deficiency-associated APOB mutation impacts lipid metabolism in Holstein calves and breeding bulls.

During the last months, the number of reports on Holstein calves suffering from incurable idiopathic diarrhea dramatically increased. Affected calves showed severe hypocholesterolemia and mostly died within days up to a few months after birth. This new autosomal monogenic recessive inherited fat metabolism disorder, termed cholesterol deficiency (CD), is caused by a loss of function mutation of the bovine gene. The objective of the present study was to investigate specific components of lipid metabolism in 6 homozygous for the mutation (CDS) and 6 normal Holstein calves with different genotypes. Independent of sex, CDS had significantly lower plasma concentrations of total cholesterol (TC), free cholesterol (FC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), triacylglycerides (TAG), and phospholipids (PL) compared with homozygous wild-type calves ( < 0.05). Furthermore, we studied the effect of the genotype on cholesterol metabolism in adult Holstein breeding bulls of Swissgenetics. Among a total of 254 adult males, the homozygous mutant genotype was absent, 36 bulls were heterozygous carriers (CDC), and 218 bulls were homozygous wild-type (CDF). In CDC bulls, plasma concentrations of TC, FC, HDL-C, LDL-C, VLDL-C, TAG, and PL were lower compared with CDF bulls ( < 0.05). The ratios of FC:cholesteryl esters (CE) and FC:TC were higher in CDC bulls compared with CDF bulls, whereas the ratio of CE:TC was lower in CDC bulls compared with CDF bulls ( < 0.01). In conclusion, the CD-associated mutation was shown to affect lipid metabolism in affected Holstein calves and adult breeding bulls. Besides cholesterol, the concentrations of PL, TAG, and lipoproteins also were distinctly reduced in homozygous and heterozygous carriers of the mutation. Beyond malabsorption of dietary lipids, deleterious effects of apolipoprotein B deficiency on hepatic lipid metabolism, steroid biosynthesis, and cell membrane function can be expected, which may result in unspecific symptoms of reduced fertility, growth, and health.

Investigation of the low-density lipoprotein receptor gene and cholesterol as a risk factor for migraine

The Low-Density Lipoprotein Receptor (LDLR) gene is a cell surface receptor that plays an important role in cholesterol homeostasis. We investigated the (TA)n polymorphism in exon 18 of the LDLR gene on chromosome 19p13.2 performing an association analysis in 244 typical migraine-affected patients, 151 suffering from migraine with aura (MA), 96 with migraine without aura (MO) and 244 unaffected controls. The populations consisted of Caucasians only, and controls were age- and sex-matched. The results showed no significant difference between groups for allele frequency distributions of the (TA)n polymorphism even after separation of the migraine-affected individuals into subgroups of MA and MO affected patients. This is in contradiction to Mochi et al. [Mochi M, Cevoli S, Cortelli P, Pierangeli G, Scapoli C, Soriani S, Montagna P. Investigation of an LDLR gene polymorphism (19p13.2) in susceptibility to migrane without aura. J Neurol Sci 2003; 213 (1-2): 7-10.] who found a positive association of this variant with MO. Our study discusses possible differences between the two studies and extends this research by investigating circulating cholesterol levels in a migraine-affected population. (C) 2004 Elsevier B.V. All rights reserved.

Plasma delipidation process induces rapid regression of atherosclerosis and mobilisation of adipose tissue

Cholesterol is a major component of atherosclerotic plaques. Cholesterol accumulation within the arterial intima and atherosclerotic plaques is determined by the difference of cellular cholesterol synthesis and/or influx from apo B-containing lipoproteins and cholesterol efflux. In humans, apo A-I Milano infusion has led to rapid regression of atherosclerosis in coronary arteries. We hypothesised that a multifunctional plasma delipidation process (PDP) would lead to rapid regression of experimental atherosclerosis and probably impact on adipose tissue lipids. In hyperlipidemic animals, the plasma concentrations of cholesterol, triglyceride and phospholipid were, respectively, 6-, 157-, and 18-fold higher than control animals, which consequently resulted in atherosclerosis. PDP consisted of delipidation of plasma with a mixture of butanol-diisopropyl ether (DIPE). PDP removed considerably more lipid from the hyperlipidemic animals than in normolipidemic animals. PDP treatment of hyperlipidemic animals markedly reduced intensity of lipid staining materials in the arterial wall and led to dramatic reduction of lipid in the adipose tissue. Five PDP treatments increased apolipoprotein A1 concentrations in all animals. Biochemical and hematological parameters were unaffected during PDP treatment. These results show that five PDP treatments led to marked reduction in avian atherosclerosis and removal of lipid from adipose tissue. PDP is a highly effective method for rapid regression of atherosclerosis.

Kinetic study of the thermal degradation of high density polyethylene

The thermal degradation of high density polyethylene has been modelled by the random breakage of polymer bonds, using a set of population balance equations. A model was proposed in which the population balances were lumped into representative sizes so that the experimentally determined molecular weight distribution of the original polymer could be used as the initial condition. This model was then compared to two different cases of the unlumped population balance which assumed unimolecular initial distributions of 100 and 500 monomer units, respectively. The model that utilised the experimentally determined molecular weight distribution was found to best describe the experimental data. The model fits suggested a second mechanism in addition to random breakage at slow reaction rates. (c) 2005 Elsevier Ltd. All rights reserved.