High intralysosomal levels of lipoprotein cholesterol influence the endocytic activity and acid hydrolase content of CH-23 fibroblasts

Lipoprotein extracts isolated from hyperlipemic rabbit serum were used to study the effects of abnormal lipid levels on the functions of the lysosomal system in Chinese hamster CH-23 fibroblasts. The ability of cells enriched with lipid to endocystose [³H]inulin was largely unimpaired and utilising density gradient fractionation procedures the fusion between incoming inulin and low density lysosomes ladened with esterified cholesterol could be demonstrated. Studies in which cells were exposed to short ‘burst’ of [³H]inulin indicated, however, that the rate of fusion between inulin and secondary lysosomes was reduced. The incorporation of lipid material also produced a rapid though transient increase in several acid hydrolase activities. The stimulus for increased enzyme activity does not appear to be the deposition of ingested lipid within the lysosomes but rather at some stage prior to this, possibly the formation of endocytic vesicles. The current findings suggest that the intralysosomal incorporation of lipid material which occurs in several pathological conditions has marked effects on lysosomal function.     

Effects of part sequences of human growth hormone on in vivo hepatic glycogen metabolism in the rat

Acute effects of two part sequences of human growth hormone on the in vivo activity levels of hepatic glycogen synthase and glycogen phosphorylase were examined. The peptide corresponding to residues 6 to 13 of the hormone (hGH 6–13) decreased the percentage of phosphorylase in the active form without affecting synthase activity. This action was indirect and dependent upon insulin. The peptide hGH 177–191 decreased the level of the active form of synthase without affecting phosphorylase activity. This effect was also observed with analogous peptides containing the sequence hGH 178–191 (i.e., hGH 172–191 and hGH 178–191), whereas the peptide hGH 179–191 was inert.The onset of these effects was rapid, and maximum changes in activity were produced in 5 min by both peptides. The effect for hGH 177–191 was short-lived, and synthase activity had returned to normal levels by 15 min, whereas the action of hGH 6–13 was of longer duration and was still quite marked at 60 min. Both peptides showed a linear dependence of response to the log dose of peptide injected over the range 0.1–250 μg hGH 6–13 per kg body weight and 0.05–25 gmg hGH 177–191 per kg body weight. Hepatic 3′,5′-cyclicadenylic acid levels were not affected by either peptide. Incorporation of glycerol carbon liver glycogen was increased by hGH 6–13 and decreased by hGH 177–191. This discussed in terms of a futile cycle between glycogen and hexone phosphate in the liver, as the basis for a control mechanism for hepatic glycogen metabolism. The present observations are consistent with other in vivo and in vitro actions of these and related peptides.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Synthesis,processing, and secretion of hepatic very low density lipoprotein

Very low density lipoprotein (VLDL) is the major vehicle in the plasma which carries triacylglycerol synthesized in the liver to peripheral tissues for utilization. Estrogen-induced chick parenchymal liver cells (hepatocytes) synthesize and secrete large amounts of VLDL. These cells, in a primary monolayer culture system developed in this laboratory, have been employed to study the operative and regulatory aspects of VLDL synthesis, assembly, and secretion. Some 10 min are required for the translation of the principle VLDL protein constituent, apolipoprotein B, and 30–35 min are required for the two newly translated chick VLDL apolipoproteins, apolipoprotein B and apolipoprotein II, to be secreted. Apolipoprotein B is synthesized on membrane-bound polysomes as a contiguous polypeptide chain of 350K molecular weight (MW) and is not assembled posttranslationally from smaller-peptide precursors. Translocation of puromycin-discharged apolipoprotein B nascent chains into the endoplasmic reticulum lumen and their subsequent secretion are independent of both ongoing protein synthesis and the attachment of the nascent peptides to ribosomes. Apolipoprotein B nascent chains discharged by puromycin assemble with glycerolipid (mainly triacylglycerol) and are secreted as immunoprecipitable VLDL. Core oligosaccharides are added to the apolipoprotein B nascent chain co-translationally in at least two stages, at molecular weights of ～ 120K and ～ 280K. Inhibition of N-linked glycosylation of apolipoprotein B with tunicamycin affects neither the assembly of glycerolipids into VLDL nor the secretion of the VLDL particle, indicating that aglyco-apolipoprotein B can serve as a functional component for VLDL assembly and secretion. Active synthesis of the VLDL apolipoproteins is required, however, for glycerolipid assembly into VLDL and secretion from the hepatocyte. The differential kinetics with which newly synthesized apolipoproteins and glycerolipids are secreted as VLDL and the timing of the effects of protein-synthesis inhibitors on their secretion indicate that VLDL constituents are assembled sequentially in the intact liver cell. The bulk of the VLDL triacylglycerol and some VLDL phosphoglyceride is introduced early in the secretory pathway proximal, yet subsequent to apopeptide synthesis, while a significant fraction of VLDL phosphoglyceride associates with the resulting triacylglycerol-rich lipid-protein complexes just prior to their secretion as mature VLDL. Within the context of current models for VLDL structure, the late assembly of phosphoglyceride into VLDL is taken to represent a surface maturation of the nascent VLDL particle.     

Low density lipoprotein (LDL), atherosclerosis and antioxidants

A crucial and causative role in the pathogenesis of atherosclerosis is believed to be the oxidative modification of low density lipoprotein (LDL). The oxidation of LDL involves released free radical driven lipid peroxidation. Several lines of evidence support the role of oxidized LDL in atherogenesis. Epidemiologic studies have demonstrated an association between an increased intake of dietary antioxidant vitamins, such as vitamin E and vitamin C and reduced morbidity and mortality from coronary artery diseases. It is thus hypothesized that dietary antioxidants may help prevent the development and progression of atherosclerosis. The oxidation of LDL has been shown to be reduced by antioxidants, and, in animal models, improved antioxidants may offer possibilities for the prevention of atherosclerosis. The results of several on going long randomized intervention trials will provide valuahle information on the efficacy and safety of improved antioxidants in the prevention of atherosclerosis. This review a evaluates current literature involving antioxidants and vascular disease, with a particular focus on the potential mechanisms.     

Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR-/-) mice. Collectively, although VLDLR deficiency in LRP- and LDLR-/- mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.     

Antioxidative activity on human low density lipoprotein (LDL) oxidation by pentagalloic acid

The aim of this study was to investigate the efficiency of the pentagalloic acid compound in inhibiting the metal ions and cell lines that mediate in low density lipoprotein (LDL) oxidation. Pentagalloic acid prolonged the lag time preceeding the onset of conjugated diene formation. In chemically induced LDL oxidation by Cu²⁺ plus hydrogen peroxide or peroxyl radical generated by 2, 2′-azo-bis (2-amidino propane) hydrochloride (AAPH), pentagalloic acid inhibited LDL oxidation as monitored by measuring the thiobarbituric acid reactive substances (TBARS), malondialdehyde (MDA), and gel electrophoretic mobility. The physiological relevance of the antioxidative activity was validated at the cellular level where pentagalloic acid inhibited mouse macrophage J774 and endothelial cell-mediated LDL oxidation. When compared with several other antioxidants, pentagalloic acid showed a much higher ability than naturally occuring antioxidants, α-tocopherol and ascorbic acid, and the synthetic antioxidant, probucol.     

一次密度梯度超速离心分离人血清的VLDL、LDL、HDL_2、HDL_3及无脂血清

本文将密度梯度、离心力和离心时间作适当的组合和配比:即不连续密度梯度1.000—1,400g/mlNaBr溶液、离心力168,000g、22小时,10℃,在Beckman L8-80型、区带头Ti-14一次超速离心将血清中四种主要脂蛋白和无脂血清相互分开,获得五个明显的蛋白峰。前四个峰经琼脂糖电泳,聚丙烯酰胺电泳,免疫双扩散和分析超速离心鉴定分别为VLDL、LDL、HDL_2和HDL_3,不含清蛋白。峰五为无脂血清,仅含0.046mg/dl胆固醇和0.2mg/dl甘油三酯,本法重复性佳,分离样品多(50ml),效果好,操作简单,并可延长离心机头的使用期限。已用于研究各种因素对脂蛋白含量的影响及其代谢间的相互关系。     

Overexpression of apolipoprotein AV in the liver reduces plasma triglyceride and cholesterol but not HDL in ApoE deficient mice

It has been shown that adenovirus-mediated overexpression of human ApoAV (hApoAV) in C57BL/6 mice results in decreased plasma triglyceride (TG) and total cholesterol (TC) levels with a major reduction occurring in the HDL fraction. In order to study the effect of ApoAV on hypercholesterolemic mice, an adenoviral vector expressing hApoAV was constructed and injected into ApoE deficient mice. High levels of hApoAV mRNA in the liver and ApoAV proteins in the liver and plasma were detected. The treatment reduced plasma TG levels by 50% and 75%, and TC levels by 45% and 58% at day 3 and 7, respectively, after treatment as compared with a control group treated with Ad-hAP (human alkaline phosphatase). Plasma HDL-C levels remained unaltered, which were different from normolipidemic mice. These findings suggest that ApoAV might serve as a therapeutic agent for hyperlipidemic disorder.     

Up-regulation of ATP binding cassette transporter A1 expression by very low density lipoprotein receptor and apolipoprotein E receptor 2

Activation of very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (apoER2) results in either pro- or anti-atherogenic effects depending on the ligand. Using reelin and apoE as ligands, we studied the impact of VLDLR- and apoER2-mediated signaling on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux using RAW264.7 cells. Treatment of these mouse macrophages with reelin or human apoE3 significantly increased ABCA1 mRNA and protein levels, and apoAI-mediated cholesterol efflux. In addition, both reelin and apoE3 significantly increased phosphorylated disabled-1 (Dab1), phosphatidylinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ), and specificity protein 1 (Sp1). This reelin- or apoER2-mediated up-regulation of ABCA1 expression was suppressed by 1) knockdown of Dab1, VLDLR, and apoER2 with small interfering RNAs (siRNAs), 2) inhibition of PI3K and PKC with kinase inhibitors, 3) overexpression of kinase-dead PKCζ, and 4) inhibition of Sp1 DNA binding with mithramycin A. Activation of the Dab1-PI3K signaling pathway has been implicated in VLDLR- and apoER2-mediated cellular functions, whereas the PI3K-PKCζ-Sp1 signaling cascade has been implicated in the regulation of ABCA1 expression induced by apoE/apoB-carrying lipoproteins. Taken together, these data support a model in which activation of VLDLR and apoER2 by reelin and apoE induces ABCA1 expression and cholesterol efflux via a Dab1-PI3K-PKCζ-Sp1 signaling cascade.     

Resonance Raman spectra of beta-carotene in native and modified low-density lipoprotein

Low-density lipoproteins isolated between density 1.02 and 1.063 g/cm3 from normal fasting human plasma, show strong resonance Raman spectra due to the presence of beta-carotene. Three intense bands, at 1010, 1160 and 1530 cm-1, are assigned to the stretching vibrations of -C-CH3, = C-C = and -C = C- bonds, respectively, of beta-carotene. High-resolution spectra of the 1500-1600 cm-1 region reveal multiple features, suggesting the coexistence of several structural populations of beta-carotene. The modifications of lipoproteins with pH and temperature (30 degrees-42 degrees) change the resonance Raman spectra of beta-carotene. The specific binding of LDL at pH 7.0 by fibroblast cells is suppressed. Our experiments thus suggest that physical and chemical perturbations of plasma lipoproteins modify the lipid-protein interactions and thereby alter the configurational distribution of beta-carotene molecules within these particles.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of ¹²⁵I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on ¹²⁵I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of ¹²⁵I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

Apolipoprotein E gene polymorphisms in Lebanese with hypercholesterolemia

Apolipoprotein E (ApoE) has an important role in the metabolism of lipids through its major isoforms (ε2, ε3, ε4). In particular, ApoE ε4, has been considered as a major genetic risk factor for cardiovascular diseases (CVD). The aim of our study is to investigate the frequency of ApoE gene polymorphisms (rs 429358C > T, rs 7412C > T) and their relationship to lipid parameters in a group of Lebanese hypercholesterolemic subjects (22 males and 24 females, aged 25–80 years). Lipid profile, apolipoproteins A-I and B were determined using fasting serum samples; and molecular analysis of ApoE polymorphisms using blood in EDTA tubes. The distribution of the four ApoE genotypes detected in this study was: ε3/ε3 (73.9%), ε3/ε4 (17.4%), ε2/ε3 (6.5%), and ε2/ε4 (2.2%) resulting in allelic frequencies for ε2, ε3 and ε4 of 4.3%, 85.9% and 9.8%, respectively. No association was determined among any of the lipid parameters, gender and ApoE genotypes. Lipid parameters were not statistically different among various ApoE genotypes (p > 0.05). ApoE ε2 frequency was found to be lower than that previously reported for healthy Lebanese (7.2%). CVD is one of the major leading causes of mortality in Lebanon with a reported prevalence of 12.2% in males and 7.7% in females, which incidentally agrees with our finding regarding ε4 allelic frequency of 13.6% in males and 6.3% in females. Consequently, larger prospective studies are recommended to highlight the correlation of ApoE polymorphisms to other biochemical and environmental factors involved in CVD.     

CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu(2+)-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.     

Effect of Licorice Flavonoid Oil on Cholesterol Metabolism in High Fat Diet Rats

Dietary licorice fravonoid oil (LFO) significantly decreased hepatic cholesterol and plasma lipoprotein cholesterol levels in high-fat diet rats. It significantly suppressed hydroxymethylglutaryl-CoA synthase activity and increased cholesterol 7α-hydroxylase activity. The low density lipoprotein receptor mRNA level was significantly increased by LFO. These results suggest that dietary LFO improves cholesterol metabolism in obese animals.     

Molecular structure of low density lipoprotein: current status and future challenges

This review highlights recent advances in structural studies on low density lipoprotein (LDL) with particular emphasis on the apolipoprotein moiety of LDL, apolipoprotein B100 (apoB100). Various molecular aspects of LDL are outlined and obstacles to structure determination are addressed. In this context, the prevailing conceptions of the molecular assembly of LDL and how the synergy of complementary biochemical, biophysical and molecular simulation approaches has lead to the current structural model of LDL are discussed. Evidence is presented that structural heterogeneity and the intrinsic dynamics of LDL are key determinants of the functionality of LDL in both health and disease. Some key research directions, remaining open questions and rapidly emerging new concepts for medical applications of LDL, are furthermore outlined. The article concludes by providing an outlook concerning promising future strategies for the clarification of the molecular details of LDL, in particular of apoB100, combining recent advances in molecular modeling with developments of novel experimental techniques. Although new insights into the molecular organization of LDL are forthcoming, many open questions remain unanswered. The major challenge of the next decade will certainly be the elucidation of the molecular structural and dynamic features of apoB100.     

Preferred orientations of LDL in vitreous ice indicate a discoid shape of the lipoprotein particle

The structure of the human low-density lipoprotein (LDL) was analyzed in vitreous ice using cryo-electron microscopy (cryo-EM). In relatively thick cryo-EM preparations, random orientation of LDL particles produced various types of projections on the microscope screen, including circular projections with a high-density ring and rectangular projections with two high-density bands. However, in especially thin preparations, preferred, non-random orientations of the LDL particle produced only circular projections of the lipoprotein structure. In preparations with high LDL concentrations, ordered two-dimensional arrays, including hexagonal arrangements of circular projections and short stacks of rectangular projections, were observed. These observations are consistent with a discoid shape of the LDL particle, and suggest that surface tension forces may influence orientation of the LDL disc in thin aqueous films. Face-on orientation of LDL in especially thin cryo-EM preparations may explain earlier difficulties in identifying discoid features of the lipoprotein particle, and illustrates that some caution is warranted when attempts are made to reconstruct the three-dimensional structure of LDL from cryo-electron micrographs.     

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.