Effect of thyroid hormones on acid cholesterol ester hydrolase activity in rat liver,heart and epididymal fat pads

The regulation of acid cholesterol ester hydrolase activity by thyroid hormones was studied in subcellular fractions from rat liver, heart, and epididymal fat pads; hydrolase activity was determined at pH 5 with a glycerol-dispersed cholesterol oleate substrate preparation. Acid cholesterol ester hydrolase activity was decreased in liver preparations from thyroidectomized rats relative to activity in livers from euthyroid control rats. Administration of triidothyronine to either euthyroid or hypothyroid (thyroidectomized) rats resulted in an increase in acid cholesterol ester hydrolase activity in liver preparations. Similar effects of thyroidectomy and the administration of triiodothyronine on acid cholesterol ester hydrolase activity were observed with fat pad preparations. In contrast, no effect of thyroid hormones was observed on acid cholesterol ester hydrolase activity in heart. These results suggest that thyroid hormones may regulate the catabolism of serum lipoproteins, in part, by alterations in lysosomal acid cholesterol ester hydrolase activity in liver and epididymal fat pads.     

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.     

Rates of cholesterol exchange between human erythrocytes and plasma lipoproteins

Rates of exchange of labelled cholesterol between human erythrocytes and three plasma lipoprotein species, LDL, HDL₂ and HDL₃, were measured over a range of lipoprotein concentrations and temperatures. The exchange rates reached limiting, concentration-independent values, which were the same for the three lipoproteins. The temperature dependencies correspond to activation energies of 12 kcal in the limiting rate region, and at lower lipoprotein concentrations to activation energies of 11 to 22 kcal. Calculations based on simple collision theory indicate that energetic barriers to the exchange are far smaller than indicated by these activation energies and that no particular orientation of lipoprotein molecules is required for the exchange. The occurrence of a limiting rate may be a result of the adsorption of lipoprotein molecules onto a limited number of sites on the cell surface, or of a slow process occurring in the membrane, possibly the diffusion of cholesterol. Present data do not permit a choice between these models.     

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.     

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.     

Comparative study of Ca2+ binding to lipoprotein(a) and low density lipoprotein by spin labeling

The effect of Ca2+ binding on the dynamic properties of various spin labeled fatty acids in lipoprotein(a) (Lp(a)) was studied in comparison with low density lipoprotein (LDL) isolated from human plasma. In contrast to LDL, binding of Ca2+ to Lp(a) induced broadening of the lines in the ESR spectra of the spin labeled stearic acids. In 1.6 M NaBr solutions the thermotropic change in the surface structure was observed in both lipoproteins at similar temperatures. Ten millimolar concentration of Ca2+ shifted the temperature of the thermotropic change in the surface structure of Lp(a) to considerably higher values. We conclude that Ca2+ binding to Lp(a) induces changes in the lipid structure of the particle surface.     

In vivo study of free and esterified cholesterol turnover in various tissues of the rat.

Rats were infused for 3.5 to 10 hrs with either red cells or plasma previously labelled in vivo by [3H]-cholesterol. Cholesterol specific radioactivities were measured in plasma, HDL, LDL and VLDL, and various tissues. Red cell infusions led to a higher labelling of free than of esterified cholesterol in the plasma of infused rats. The opposite situation was observed following plasma infusion. Comparison of free and esterified cholesterol specific radioactivities in each tissue showed that esterified cholesterol was transferred from plasma to all the tissues, except the adrenals. Study of the ratios of cholesterol specific radioactivities from one experimental group to the other in each tissue, made it possible to demonstrate clearly the occurence of hydrolysis within all the studied tissues except 5 of them where its existence remains uncertain (lung, heart, kidney, tendon, muscle) and of esterification in 3 tissues (adrenal, liver lung). In addition, ratios of cholesterol radioactivities (free/ester) were found to be identical in plasma and in 4 tissues, where neither hydrolysis nor esterification were detected (heart, muscle, kidney, tendon). This finding is an argument in favor of a simultaneous transport of free and esterified cholesterol from plasma into these 4 tissues and suggests that the entire lipoprotein particles can penetrate these tissues, with no specificity of one special class. In adrenal, unlike all other tissues: 1) the turnover of esterified cholesterol was achieved mostly by hydrolysis and esterification in situ; 2) a preferential lipoprotein class (LDL) was responsible for the transport of free cholesterol from the plasma.     

Distinct functional domains contribute to degradation of the low density lipoprotein receptor (LDLR) by the E3 ubiquitin ligase inducible Degrader of the LDLR (IDOL)

We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR). We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL. The RING domain promotes ubiquitination in vitro and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation. We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL. The FERM domain interacts with the LDLR and in living cells co-localizes with the receptor at the plasma membrane. Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain. Mutating residues within this region or residues in the LDLR preceding the NPVY endocytosis motif abrogate LDLR degradation by IDOL. Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL. Our findings may facilitate development of structure-based IDOL inhibitors aimed at increasing LDLR abundance in therapeutic strategies to treat cardiovascular disease.     

Protective effect of the oligomeric acylphloroglucinols from Myrtus communis on cholesterol and human low density lipoprotein oxidation

Myrtle (Myrtus communis L.), a culinary spice and flavouring agent for alcoholic beverages widespread in the Mediterranean area and especially in Sardinia, contains the structurally unique oligomeric non-prenylated acylphloroglucinols, semimyrtucommulone and myrtucommulone A, whose antioxidant activity was investigated during the oxidative modification of lipid molecules implicated in the onset of cardiovascular diseases. Both acylphloroglucinols showed powerful antioxidant properties during the thermal (140 degrees C), solvent-free degradation of cholesterol. Moreover, the pre-treatment with semimyrtucommulone and myrtucommulone A significantly preserved LDL from oxidative damage induced by Cu(2+) ions at 2h of oxidation, and showed remarkable protective effect on the reduction of polyunsaturated fatty acids and cholesterol, inhibiting the increase of their oxidative products (conjugated dienes fatty acids hydroperoxides, 7beta-hydroxycholesterol, and 7-ketocholesterol). Taking into account the widespread culinary use of myrtle leaves, the results of the present work qualify the natural compounds semimyrtucommulone and myrtucommulone A as interesting dietary antioxidants with potential antiatherogenicity.     

Binding of unmodified low-density lipoproteins to human fibroblasts. An investigation by immunoelectron microscopy

The binding of unmodified low density lipoproteins to the plasma membrane of fibroblasts was studied at the ultrastructural level. The bound low density lipoprotein was visualized by an indirect immunoperoxidase technique, with the use of an antiserum against apoprotein B. Immunoreactive regions representing bound apoprotein B were found on the plasma membrane, in indented regions with a diameter of 0.15–0.30 μm and a fuzzy coat on the cytoplasmic side. Fibroblasts from a patient homozygous for hyperlipoproteinaemia type IIa showed no immunoreactive material in the indented regions. The specific ¹²⁵I-labelled low density lipoprotein binding to these homozygous fibroblasts was 7% compared to control fibroblasts.     

Molecular parameters that control the association of low density lipoprotein apo B-100 with chondroitin sulphate

The association of low density lipoprotein (LDL) with proteoglycans of the intima, in particular chondroitin 6-sulphate proteoglycans, may contribute to LDL accumulation during atherogenesis. We studied the interactions of apolipoprotein B-100 (apo B-100) peptide segments and model peptides with chondroitin 6-sulphate. The ability of these peptides to inhibit complex formation between LDL and chondroitin 6-sulphate was used as a measurement of the interaction. Results from earlier studies suggest that surface located segments of apo B-100 are responsible for the interaction of LDL with heparin and chondroitin sulphate-rich arterial proteoglycans. Therefore 16 hydrophilic apo B-100 peptides were selected for studies and synthesized with a peptide synthesizer. These synthetic peptides were 7 to 26 amino acids long. Four of the peptides inhibited the association of LDL with chondroitin 6-sulphate, namely apo B segments 4230–4254, 3359–3377, 3145–3157 and 2106–2121. The 3359–3377 segment was the most efficient. A common feature betweeb the interacting peptides was an excess of positively charged side chains and based on these results we synthesized nine model peptides that shared sequence characateristics with the interacting apo B-100 peptides. Five of these: RSGRKRSGK, RSSRKRSGK, RGGRKRGGK, RSRSRSRSR AND RGRGRGRGR were shown to block the LDL-chrondroitin-6-sulphate association, RSRSRSRSR being the most effective. The results suggest that the optimal association of the peptides with chrondroitin 6-sulphate is obtained with a minimal chain length of nine amino acids and a minimum of five positive charges and that flexibility in the binding region is important.     

The central type Y amphipathic alpha-helices of apolipoprotein AI are involved in the mobilization of intracellular cholesterol depots

We studied the role of a central domain of human apolipoprotein AI (apoAI) in cholesterol mobilization and removal from cells. In order to check different protein conformations, we tested different sized and cholesterol-content reconstituted apoAI particles (rHDL). Meanwhile cholesterol-free discs were active to induce mobilization, only small cholesterol-containing rHDL were active. To test the influence of a central domain in such events, we used two apoAI variants: one, with its central Y helix pair replaced by the C-terminal domain, and the other having a lysine deleted in central region. The helix-swapping variant decrease the cholesterol pool available to acyl-CoA cholesterol acyl transferase and increase mobilization of newly synthesized cholesterol. Instead, the deletion mutant had no effect on both events. We conclude that the central domain of apoAI is involved in cholesterol cell traffic and solubilization, and that a Y-type charge distribution in polar face may be required, as well as a correct helices-polar face orientation.     

Comparison of the intracellular pathways of immunoglobulin-G and low density lipoprotein in cultured human term trophoblast cells

Trophoblast cells were cultured on microporous membrane filters. After incubation at different times with gold-conjugated ligands, the cells were processed for electron microscopy. Gold particles indicating the presence of both IgG and LDL appeared in a time-dependent manner in coated pits and coated vesicles. LDL-gold appeared primarily within lysosomes whereas approximately 50% of the internalized IgG-gold appeared within vesicles (diameters ranging from 35 to 80 nm) near the basal regions of the cell. These vesicles may be the protective mechanism which prevents IgG breakdown during transcytosis across trophoblast cells, thus allowing transport of the intact molecule to the fetus.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Niemann-Pick Type II fibroblasts exhibit impaired cholesterol esterification in response to shingomyelin hydrolysis

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (> 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [³H]oleic acid into cholesterol-[³H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimuated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquinone abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.     

Effects of PCSK9 genetic variants on plasma LDL cholesterol levels and risk of premature myocardial infarction in the Italian population

The R46L variant in the proprotein-convertase subtilisin-kexin type 9 (PCSK9) gene was associated with reduced levels of LDL and total cholesterol and with a lower risk of coronary artery disease. We investigated the association of R46L with myocardial infarction (MI) in 1,880 Italian patients with premature MI and 1,880 controls. A trend toward a protective effect of the L46 allele was observed [odds ratio (OR) = 0.75, 95% confidence interval (CI) = 0.49–1.13; P = 0.17], although the association with MI was not significant. This is probably due to the combined effect of the low frequency of R46L among Italians and of the young age of the analyzed cohort for whom the impact of coronary atherosclerosis is less important. This hypothesis was indirectly confirmed by the significant association found after including 1,056 additional older controls (OR = 0.67, 95% CI = 0.46-0.97; P = 0.036). LDL cholesterol was significantly lower in L46 carriers (116.2 ± 34.7 mg/dl) than in noncarriers (137.4 ± 47.3 mg/dl; P = 0.00022); a similar reduction was observed for total cholesterol (191.7 ± 37.7 vs. 211.7 ± 49 mg/dl; P = 0.00019). Analysis of 23 additional polymorphisms in the PCSK9 region identified another single nucleotide polymorphism (SNP) (rs11206510) associated with cholesterol levels. We confirmed that the L46 allele not only decreases LDL cholesterol but also protects against MI. Moreover, we replicated the association of total and LDL cholesterol with the SNP rs11206510.