| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
| First Page | Previous Page | Next Page | Last Page |
| In vitro effects of selenite and mercuric chloride on liver thiobarbituric acid-reactive substances and non-protein thiols from rats: influences of dietary cholesterol and polyunsaturated and saturated fatty acids Farina, M., F. A. Soares, et al. (2003), Nutrition 19(6): 531-5. Abstract: OBJECTIVE: We measured the in vitro effects of mercuric chloride (Hg2+) and selenite (Se4+) on hepatic 2-thiobarbituric acid-reactive substances (TBARS) and non-protein sulfhydryl (NPSH) levels of rats fed diets enriched with polyunsaturated or saturated fatty acids with and without cholesterol. METHODS: Male Wistar rats (21 d old) were assigned to one of four groups and fed diets containing 20% soybean oil, 20% soybean oil plus 1% cholesterol, 20% coconut oil, or coconut oil plus 1% cholesterol. After the feeding period (6 wk), body weight gain was equal in all groups. TBARS levels and NPSH content were measured after in vitro exposure to mercuric chloride (100 microM) and sodium selenite (25 microM) for 1 h. RESULTS: The lipid peroxidation, measured as TBARS levels in the control group, were statistically higher in hepatic homogenates of rats fed diets containing soybean oil than in groups fed coconut oil (P = 0.009). However, cholesterol supplementation did not change TBARS levels. Selenite alone did not modify TBARS production, whereas mercury alone significantly increased TBARS levels. Moreover, Se4+ protected against mercury-induced lipid peroxidation only in rats fed diets containing coconut oil. In the control group, dietary fat acids did not change NPSH levels. Selenite produced higher oxidative effects toward NPSH content, whereas Hg2+ decreased NPSH levels only in liver from rats fed diets containing soybean oil. NPSH levels were higher after concomitant exposure to Se4+ and Hg2+ chloride that after exposure to Se4+ alone, suggesting an interaction between Hg2+ and Se4+. Catalase activity was higher in animals fed diets containing soybean oil. Dietary cholesterol decreased glutathione peroxidase activity. CONCLUSION: Together these results indicated that the protective effect of Se4+ against mercury-induced lipid peroxidation depends on dietary fat saturation. |
| In vitro expression of structural defects in the lecithin-cholesterol acyltransferase gene Klein, H. G., N. Duverger, et al. (1995), J Biol Chem 270(16): 9443-7. Abstract: Classic LCAT deficiency (CLD) and fish eye disease (FED) are two clinically distinct syndromes, associated with defects in the lecithin-cholesterol acyltransferase (LCAT) gene resulting in total (CLD) or partial (FED) enzyme deficiency. In order to investigate the underlying molecular mechanisms that lead to different phenotypic expression in CLD and FED, LCAT mutants associated with either CLD (LCAT147, LCAT156, and LCAT228) or FED (LCAT10, LCAT123, LCAT158, LCAT293, LCAT300, and LCAT347) were expressed in vitro in human embryonic kidney 293 cells and characterized with respect to LCAT expression and enzyme activity. Evaluation of mutant LCAT gene transcription by Northern blot analysis demonstrated LCAT mRNA of normal size and concentration. Although all constructs gave rise to similar intracellular LCAT mass, the amount of enzyme present in the media for LCAT147, LCAT156, and LCAT300 was reduced to less than 10% of normal, suggesting that these mutations disrupted LCAT secretion. Western blot analysis of cell culture media containing wild type or mutant LCAT demonstrated the presence of a single normal-sized band of 67 kDa. The ability of the different enzymes to esterify free cholesterol in high density lipoprotein-like proteoliposomes (alpha-LCAT-specific activity) was reduced to less than 5% of normal for CLD mutants LCAT147 and LCAT228 and FED mutants LCAT10, LCAT123, LCAT293, and LCAT347, whereas that of LCAT156, LCAT158, and LCAT300 ranged from 45 to 110% of control. Although most FED mutant LCAT enzymes retained the ability to esterify free cholesterol present in alpha- and beta-lipoproteins of heat-inactivated plasma, esterification was undetectable in all CLD mutants (LCAT147, LCAT156, and LCAT228). In contrast, all mutant enzymes retained the ability to hydrolyze the water soluble, short-chained fatty acid substrate p-nitrophenolbutyrate. In summary, our studies establish the functional significance of nine LCAT gene defects associated with either FED or CLD. Characterization of the expressed LCAT mutants identified multiple, overlapping functional abnormalities that include defects in secretion and/or disruption of enzymic activity. All nine LCAT mutants retained the ability to hydrolyze the water-soluble PNPB substrate, indicating intact hydrolytic function. Based on these studies we propose that mutations in LCAT residues 147, 156, 228 (CLD) and 10, 123, 158, 293, 300, and 347 (FED) do not disrupt the functional domain mediating LCAT phospholipase activity, but alter structural domains involved in lipid binding or transesterification. |
| In vitro fibrillogenesis of the amyloid beta 1-42 peptide: cholesterol potentiation and aspirin inhibition Harris, J. R. (2002), Micron 33(7-8): 609-26. Abstract: Understanding the formation of extracellular amyloid neurofibrillar bundles/senile plaques and their role in the development of Alzheimer's disease is of considerable interest to neuroscientists and clinicians. Major components of the extracellular neurofibrillar bundles are polymerized amyloid beta (Abeta) peptides (1-40), (1-42) and (1-43), derived in vivo from the soluble amyloid precursor protein (sAPP) by proteolytic (beta- and gamma-secretase) cleavage. The Abeta(1-42) peptide is widely considered to be of greatest significance in relation to the pathogenesis of Alzheimer's disease. A well-defined ultrastructural characteristic within Alzheimer dense plaques is the presence of helical fibrils that are believed to consist of polymerized amyloid beta, together with other associated proteins such as the serum amyloid P protein, apolipoprotein E isoform epsilon 4, alpha1-anti-chymotrypsin, catalase, glycoproteins, proteoglycans, cholesterol and other lipids. The spontaneous in vitro fibrillogenesis of chemically synthesized Abeta(1-42) peptide (rat sequence), following 20h incubation at 37 degrees C, has been assessed from uranyl acetate negatively stained specimens studied by transmission electron microscopy (TEM). Amyloid beta(1-42) peptide fibrillogenesis in the presence of cholesterol has been investigated using aqueous suspensions of microcrystalline cholesterol and cholesteryl acetate, globular particles of cholesteryl oleate, a soluble (micellar) cholesterol derivative (polyoxyethyl cholesteryl sebacate/cholesteryl PEG 600 sebacate), cholesterol-sphingomyelin liposomes and sphingomyelin liposomes. In all these cases, with the exception of cholesteryl oleate, considerable potentiation of long smooth helical fibril formation occurred, compared to 20h 37 degrees C control samples containing the Abeta(1-42) peptide alone. The binding of polyoxyethyl cholesteryl sebacate micelles to helical Abeta fibrils/filaments and the binding of fibrils to the surface of cholesterol and cholesteryl acetate microcrystals, and to a lesser extent on cholesteryl oleate globules, indicates an affinity of the Abeta peptide for cholesterol. This potentiation of Abeta(1-42) polymerization is likely to be mediated at the molecular level via hydrophobic interaction between the amino acid side chains of the peptide and the tetracyclic sterol nucleus. Addition of cupric sulphate (0.1mM) to the Abeta solution produced large disorganized fibril aggregates. Inclusion of 1mM aspirin (sodium acetylsalicylate) in the Abeta peptide alone and as an addition to Abeta peptide solution containing cholesterol, cholesteryl acetate, soluble cholesterol, sphingomyelin and sphingomyelin-cholesterol liposomes, and to 0.1mM cupric sulphate solution, completely inhibited fibrillogenesis. Instead, only non-crystalline diffuse, non-filamentous microaggregates of insoluble Abeta particles were found, free and attached to the sterol particles. The in vitro system presented here provides a way to rapidly monitor at the structural/TEM level other compounds (e.g. chelating agents, drugs, beta-sheet breaking peptides and anti-oxidants) for their effects on amyloid beta peptide fibrillogenesis (and on preformed fibril disassembly) in parallel with in vitro biochemical studies and in vivo studies using animal models of Alzheimer's disease as well as studies on man. |
| In vitro inhibitory effect of lercanidipine on cholesterol accumulation and matrix metalloproteinases secretion by macrophages Canavesi, M., N. Baldini, et al. (2004), J Cardiovasc Pharmacol 44(4): 416-22. Abstract: Plaque rupture and thromboembolism play a major role in atherosclerotic acute syndrome. Experimental studies have demonstrated the potential direct anti-atherosclerotic effects of calcium antagonists. We investigated the in vitro effect of lercanidipine (REC 15/2375), a third-generation, highly lipophilic calcium antagonist on cholesterol metabolism and matrix metalloproteinases secretion in macrophages, two functions that predispose plaques to rupture. Lercanidipine (10(-6)-10(-5) M) inhibited cholesterol esterification in macrophages and reduced cellular free and esterified cholesterol accumulation from acetylated LDL (63%, 62% of control P < 0.05, respectively). In addition, lercanidipine inhibited the release of metalloproteinases in the extracellular medium (50% and 95% inhibition at 10(-5) M for MMP-9 and MMP-2, respectively). Experiments performed with lercanidipine enantiomers or other dihydropyridine derivatives, endowed with different lipophilicity and affinity for calcium channels, indicated that the above effects could be related to the lipophilic, but not to the calcium channel blocking properties of these molecules. When cells, after exposure to the drug, were allowed to equilibrate, lercanidipine inhibitory action could be observed at initial concentrations as low as 10(-9) M, which is the actual concentration range observed in plasma in clinical settings. In conclusion, our data indicate that lercanidipine may exert potent anti-atherosclerotic effects by inhibiting macrophage functions involved in plaque stability. |
| In vitro modulation of rat adipocyte ghost membrane fluidity by cholesterol oxysterols Lau, W. F. and N. P. Das (1995), Experientia 51(7): 731-7. Abstract: The effects of cholesterol and cholesterol-derived oxysterols (cholestanone, cholestenone, coprostanone and epicoprostanol) on adipocyte ghost membrane fluidity were studied using a fluorescence depolarization method. The fluorescence anisotropy of the treated membranes was determined using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Cholestanone and cholesterol decreased membrane fluidity at both the concentrations tested (10 & 50 microM) while the rest of the sterols did not exert any significant effect on membrane fluidity. In the presence of epinephrine, cholestanone partitioned more towards the lipid core but cholesterol partitioning was not affected. The fusion activation energies (delta E) obtained for membranes preincubated with cholestanone (8.6 kcal/mol) and cholesterol (8.2 kcal/mol) were not significantly different from that of untreated membranes (8.3 kcal/mol). Membranes preincubated with cholestanone and cholesterol did not exhibit any change in lipid phase throughout the temperature range (10-45 degrees C) tested. The sterols were found to inhibit fisetin-induced phospholipid methylation in isolated rat adipocytes in the rank order of cholesterol > epicoprostanol > cholestanone = cholestenone = coprostanone, while basal methylation was unaffected. When adipocytes were preincubated with the sterols before the addition of fisetin, cholestanone and cholestenone showed 74% and 66% inhibition of maximal methylation respectively. These results indicated that cholesterol oxysterols interact differently with rat adipocyte membranes, with cholestanone interacting more with phospholipids located at the inner lipid bilayer (e.g. phosphatidylethanolamine) while cholesterol interacts more with phosphatidylcholine located at the outer lipid bilayer. This differential interaction may cause selective changes in membrane fluidity at different depths of the bilayer and thus may modulate the activities of membrane-bound proteins such as enzymes and receptors. |
| In vitro oxidation of vitamin E, vitamin C, thiols and cholesterol in rat brain mitochondria incubated with free radicals Vatassery, G. T., W. E. Smith, et al. (1995), Neurochem Int 26(5): 527-35. Abstract: The kinetics of oxidation of endogenous antioxidants such as vitamins C and E and thiols as well as membrane cholesterol in isolated rat brain mitochondria were studied. Oxidation was induced by incubating the mitochondria at 37 degrees C with the free radical generators 2,2' azobis (2'-amidinopropane) dihydrochloride (ABAPH) and 2,2' azobis (2,4-dimethyl) valeronitrile (ABDVN) which undergo thermal decomposition to yield free radicals. An approximate order for the in vitro ease of oxidation was: ascorbate >> alpha-tocopherol > sulfhydryls >> cholesterol. However, small amounts of ascorbate were present in the mitochondria when alpha-tocopherol and sulfhydryl compounds were getting oxidized. This observation is different from those with more homogeneous biological substrates like blood plasma or serum. The order of oxidation of the various compounds is a function of not only the redox potentials but also the (a) concentrations of the oxidized and reduced species, (b) compartmentation of the compounds and (c) enzymatic and nonenzymatic systems for the repair or regeneration of the individual antioxidants. Even though ascorbate levels are quite low within mitochondria this nutrient may play a major role as a first line of defense against oxidative stress. The lipid-soluble ABDVN was much more potent in oxidizing membrane alpha-tocopherol and thiols than the water-soluble ABAPH. With both free radical generators the rate of oxidation of the antioxidants consisted of two phases. The initial phase, that is more rapid, may represent a pool of antioxidant that is involved in immediate antioxidant protection of the organelle with the slower compartment being responsible for replenishing the faster pool whenever needed.(ABSTRACT TRUNCATED AT 250 WORDS) |
| In vitro oxidation of vitamins C and E, cholesterol, and thiols in rat brain synaptosomes Vatassery, G. T. (1995), Lipids 30(11): 1007-13. Abstract: Free radical-induced oxidation of vitamins C, E, sulfhydryl compounds, and cholesterol in brain synaptosomes from Fisher 344 rats was studied. The synaptosomes were incubated at 37 degrees C with 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), which undergoes thermal decomposition to yield free radicals. After incubation, the synaptosomes were sedimented, saponified, and extracted with hexane to isolate tocopherol and cholesterol. Ascorbate and tocopherol were assayed by liquid chromatography, cholesterol by gas chromatography, and total sulfhydryls by spectrophotometry. Under the in vitro conditions used in this study, the approximate order for the ease of oxidation of the various compounds was: ascorbate >>tocopherol > sulfhydryl compounds >>> cholesterol. However, tocopherol and sulfhydryl oxidation occurred even before all of the ascorbate had been consumed. Therefore, the fate of a specific antioxidant at a particular cellular location cannot be predicted with complete accuracy using the in vitro order for ease of oxidation shown here. Ascorbate may play a major role in protecting brain against oxidative damage because: (i) ascorbate concentration is high in brain, (ii) it can regenerate vitamin E from its radical oxidation product, and (iii) it is one of the first antioxidants to be consumed during oxidative reactions. |
| In vitro reverse cholesterol transport from THP-1-derived macrophage-like cells with synthetic HDL particles consisting of proapolipoprotein A1 or apolipoprotein A1 and phosphatidylcholine Westman, J., C. Roobol, et al. (1995), Scand J Clin Lab Invest 55(1): 23-33. Abstract: The human monocytic leukaemia cell line THP-1 was induced to differentiate to macrophage-like cells by the addition of phorbol myristoyl acetate (PMA). Subsequently, the cells were enriched in cholesterol and these cholesterol laden cells were used to study the capability of reconstituted discoidal complexes (RDCs), consisting of either human apolipoprotein A1 (apo A1) or recombinant human proapolipoprotein A1 (proapo A1) and phosphatidylcholine (PC), to promote cholesterol efflux. RDCs containing apo A1 and proapo A1 were both effective in the mobilization of intracellular cholesterol, whether this was measured by intracellular cholesterol mass or by the appearance of radiolabelled cholesterol in the supernatant. Using the radiolabelling technique, the activity was saturable and followed Michaelis-Menten kinetics. For both types of complexes and for native HDL the maximum rate of cholesterol removed was approximately 0.5 nmol h-1 per 10(6) cells. For RDCs of proapo A1 and apo A1 and for native HDL the Km values were 3.7, 2.9 and 64.8 micrograms ml-1 respectively. A significant in vitro cholesterol efflux could only be achieved with protein-lipid complexes; no significant export was observed with either free proapo A1 or multilamellar PC liposomes without apolipoprotein. Both RDCs were found to be more active in the mobilization of intracellular cholesterol than HDL isolated from human plasma. The combined results demonstrate that synthetic complexes consisting either of apo A1 or proapo A1 and PC are both active in the in vitro reverse transport of cholesterol. |
| In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding Delavoie, F., H. Li, et al. (2003), Biochemistry 42(15): 4506-19. Abstract: Peripheral-type benzodiazepine receptor (PBR) is an 18 kDa high-affinity drug ligand and cholesterol binding protein involved in various cell functions. Antisera for distinct PBR areas identified immunoreactive proteins of 18, 40, and 56 kDa and occasionally 72, 90, and 110 kDa in testicular Leydig and breast cancer cells. These sizes may correspond to PBR polymers and correlated to the levels of reactive oxygen species. Treatment of Leydig cells with human chorionic gonadotropin rapidly induced free radical, PBR polymer, and steroid formation. UV photoirradiation generates ROS species, which increased the size of intramembraneous particles of recombinant PBR reconstituted into proteoliposomes consistent with polymer formation, determined both by SDS-PAGE and by freeze-fracture electron microscopy. Spectroscopic analysis revealed the formation of dityrosines as the covalent cross-linker between PBR monomers. Moreover, photoirradiation increased PK 11195 drug ligand binding and reduced cholesterol binding capacity of proteoliposomes. Further addition of PK 11195 drug ligand to polymers increased the rate of cholesterol binding. These data indicate that reactive oxygen species induce in vivo and in vitro the formation of covalent PBR polymers. We propose that the PBR polymer might be the functional unit responsible for ligand-activated cholesterol binding and that PBR polymerization is a dynamic process modulating the function of this receptor in cholesterol transport and other cell-specific PBR-mediated functions. |
| In vivo and in vitro studies on the regulatory link between 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in rat liver Boll, M., L. W. Weber, et al. (1999), Z Naturforsch C 54(5-6): 371-82. Abstract: The activities of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoA reductase; EC 1.1.1.34), rate-limiting enzyme of cholesterol biosynthesis, and cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), key enzyme of the neutral bile acid synthesis pathway, were measured in the microsomal fraction of rat liver and in rat liver cells to investigate the coordinate regulation of the two pathways. Both enzyme activities exhibited the same diurnal rhythm and responded in a coordinate fashion to fasting or bile acid-feeding (decrease) and to cholestyramine-feeding (increase). Cholesterol-feeding decreased the activity of HMGCoA reductase, increased that of cholesterol 7 alpha-hydroxylase, and concomitantly increased free cholesterol in microsomes. In an ex vivo setting using primary hepatocytes from animals fed a high cholesterol diet the activity of HMGCoA reductase was initially low and that of cholesterol 7 alpha-hydroxylase was elevated. Release of cholesterol into the medium with ongoing incubation caused HMGCoA reductase activity to increase, and that of cholesterol 7 alpha-hydroxylase to decline. Incubation of hepatocytes with a cholesterol-containing lipoprotein fraction stimulated the activity of cholesterol 7 alpha-hydroxylase, but left HMGCoA reductase activity unaffected. The results confirm the idea of a joint regulation of the two key enzymes of cholesterol metabolism in response to the levels of substrate and metabolites, and support the notion that with respect to bile acid and cholesterol levels, respectively, regulation of HMGCoA reductase activity may be secondary to that of cholesterol 7 alpha-hydroxylase. The in vitro studies supply evidence that the effects of cholesterol and bile acid excess or deficiency are direct and do not involve accessory changes of hormone levels or mediators. |
| In vivo biosynthesis of cholesterol in the rat retina Fliesler, S. J., R. Florman, et al. (1993), FEBS Lett 335(2): 234-8. Abstract: Previous reports have suggested that the rate of de novo cholesterol synthesis in the adult vertebrate retina is extremely slow. We investigated cholesterol biosynthesis in the adult rat retina in vivo, following intravitreal injection of 3Hacetate. HPLC analysis of retinal non-saponifiable lipid extracts revealed co-elution of radioactivity with endogenous cholesterol mass within 4.5 h post-injection. Incorporation of 3Hacetate into cholesterol was markedly reduced by co-injection of known inhibitors of the cholesterol pathway. In contrast to previous results with retinas from other species, no radiolabel or mass corresponded to squalene, except in lipid extracts from retinas treated with NB-598, a squalene epoxidase inhibitor. These results demonstrate, for the first time, the capacity of the adult vertebrate retina to rapidly synthesize cholesterol de novo. |
| In vivo blockade of tumor necrosis factor-alpha in cholesterol-fed rabbits after cardiac transplant inhibits acute coronary artery neointimal formation Clausell, N., S. Molossi, et al. (1994), Circulation 89(6): 2768-79. Abstract: BACKGROUND: We previously identified in piglet cardiac allografts an immunoinflammatory response in coronary arteries in which increased fibronectin regulated by interleukin-1 beta was associated with early evidence of intimal thickening. In the present study, we used rabbits to assess whether acute neointimal formation after cardiac transplantation was reduced by blockade of tumor necrosis factor (TNF)-alpha, which modulates interleukin-1 beta, or by cyclosporine A. METHODS AND RESULTS: Sixteen rabbits underwent heterotopic cardiac transplantation and were given saline, TNF-soluble receptor (sr), or cyclosporine A. In host hearts from saline- or TNFsr-treated groups, few coronary arteries (approximately 13% to 16%) had intimal thickening, whereas values were higher in the cyclosporine A-treated group (approximately 30%). In donor hearts from the saline-treated group, however, approximately 68% of vessels had intimal thickening versus approximately 32% in TNFsr- and approximately 30% in cyclosporine A-treated groups (P <.01 for both). Severity of intimal thickening assessed quantitatively as percent vessel area was approximately 38% in the saline-treated group but reduced in TNFsr- and cyclosporine A-treated groups to approximately 22% and 18%, respectively (P <.01 for each). Immunohistochemistry revealed increased staining for major histocompatibility complex II, T cells, interleukin-1 beta, TNF-alpha, and fibronectin in donor coronary arteries from saline-treated animals when compared with TNFsr- and cyclosporine A-treated animals. Grade 3 myocardial rejection was observed in both saline- and TNFsr-treated groups, but only grade 1 was apparent in the cyclosporine A-treated group. CONCLUSIONS: In vivo blockade of TNF-alpha suppresses the acute development of neointimal formation by selectively reducing the vascular immunoinflammatory reaction and accumulation of fibronectin, whereas cyclosporine A suppresses both the myocardial and the vascular immune reaction. |
| In vivo cholesterol kinetics in apolipoprotein E-deficient and control mice Quarfordt, S. H., B. Oswald, et al. (1995), J Lipid Res 36(6): 1227-35. Abstract: The in vivo total body cholesterol transport of homozygous apoE-deficient (-/-) and control (+/+) mice was evaluated by compartmental analysis of plasma cholesterol decay. Body cholesterol fractional catabolic rates of chow fed mutants were less (-/-, 0.17 +/- 0.02; +/+, 0.51 +/- 0.06 day-1) and body cholesterol contents greater (-/-, 68 +/- 5; +/+, 48 +/- 5 mumol) than controls. The body cholesterol expansion of the chow-fed mutant was extracellular with at least half in plasma. Cholesterol transport, i.e., the mass entering, moving through, and exiting the body each day, was similar (-/-, 6.9 +/- 0.7; +/+, 8.5 +/- 0.9 mumol/day) for homozygotes and controls on chow, and both tripled with cholesterol feeding. Differing from controls, however, mutants had considerable expansions of plasma and body cholesterol (-/-, 166 +/- 21; +/+, 59 +/- 11 mumol) with increments in peripheral tissue cholesterol contents. Cholesterol feeding increased control hepatic cholesterol without a change in plasma, whereas mutants had large increments in plasma cholesterol with no change in liver. Consistent with impaired hepatic uptake of cholesterol, mutants had much slower plasma clearance of lipoprotein cholesterol, as well as slower transfer to catabolic pools than normals. Treatment of homozygotes with lovastatin doubled both plasma cholesterol concentration and body cholesterol transport indicating the importance of apoE-dependent cell cholesterol transfer in synthetic down-regulation with this agent. These data indicate that mice lacking apoE have lower affinity hepatic uptake of plasma remnant cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS) |
| In vivo clearance of low density lipoprotein in pigeons occurs by a receptor-like mechanism that is not down-regulated by cholesterol feeding Reagan, J. W., Jr., L. R. Miller, et al. (1990), J Biol Chem 265(16): 9381-91. Abstract: The contribution of receptor-dependent and receptor-independent mechanisms for low density lipoprotein (LDL) clearance in vivo was determined in White Carneau and Show Racer pigeons fed either cholesterol free or cholesterol containing diets. The methylation of pigeon LDL resulted in the inhibition of recognition by the LDL receptor which allowed its use as a tracer of receptor-independent clearance. The fractional catabolic rate (FCR) of radiolabeled LDL in 20 control pigeons (means +/- S.E., 0.277 +/- 0.013 pools/h) was approximately seven times faster than for methylated LDL indicating that 86% of the total LDL clearance occurred by a receptor-mediated process. Total LDL clearance was reduced by 27% (FCR = 0.202 +/- 0.012 pools/h) in 14 cholesterol-fed pigeons, but receptor-mediated mechanisms were still responsible for 80% of the total LDL clearance. LDL uptake by individual tissues was measured using the residualizing label 125I-tyramine cellobiose. The liver was the primary site of LDL clearance in both control and cholesterol-fed birds. LDL receptors were active in every tissue examined and accounted for over 85% of the LDL clearance in the liver and over 90% in the adrenal gland. Consistent with the whole body LDL clearance findings, cholesterol-feeding did not significantly reduce receptor-mediated clearance of 125I-tyramine cellobiose-LDL by the liver or any of the other tissues. Hepatic sterol synthesis, however, was reduced by greater than 90% in cholesterol-fed animals. These data are consistent with the conclusion that LDL clearance in vivo in pigeons is mediated primarily by an LDL receptor-like mechanism that shows little down-regulation with hypercholesterolemia even though cholesterol synthesis is efficiently down-regulated. |
| In vivo effects of pentaerythrityl-tetranitrate and isosorbide-5-mononitrate on the development of atherosclerosis and endothelial dysfunction in cholesterol-fed rabbits Kojda, G., D. Stein, et al. (1995), J Cardiovasc Pharmacol 25(5): 763-73. Abstract: We wished to determine whether long-term treatment with organic nitrovasodilators has pharmacological effects on the development of atherosclerotic lesions and endothelial dysfunction in cholesterol-fed rabbits. For 15 weeks, six groups of 9 New Zealand White rabbits received a standard diet, which contained no admixture, pentaerythrityl-tetranitrate (PETN 6 mg/kg body weight/day), or isosorbide-5-mononitrate (ISMN 2 mg/kg body weight/day). In the other three groups, the same diets were further enriched with cholesterol (0,75%). Four rings of thoracic aorta were used for tension studies; these rings and the aortas from the aortic arch to bifurcation were then fixed in formol and stained with Sudan IV to determine the area of luminal atherosclerotic lesions by a computerized laser-scanning approach. The cholesterol diet increased plasma levels of cholesterol from 69.8 +/- 10.4 to 907.1 +/- 85.5 mg/dl. A similar result was obtained in the group receiving PETN/cholesterol, but the group fed ISMN/cholesterol showed a significantly higher plasma level of cholesterol (1,165 +/- 81.4 mg/dl). Plasma levels of PETN metabolites were still detectable by gas chromatography/mass spectrometry after a 24-h in vivo washout period. The cholesterol diet induced a pronounced degree of atherosclerotic lesions in the aortic arch and the thoracic and abdominal aorta: 73.3 +/- 1.9, 46.3 +/- 2.5, and 49.6 +/- 3.6%, respectively. Additional treatment with PETN resulted in a reduction of this atherosclerotic area to 58.6 +/- 2.05% (p < 0.0001), 34.7 +/- 1.98% (p < 0.01), and 39.3 +/- 3.06% (p < 0.05). In contrast, ISMN had no significant effect on this parameter. The cholesterol diet also induced an endothelial dysfunction as indicated by the diminished vasorelaxation induced by acetylcholine (ACh). Treatment with PETN completely inhibited the development of endothelial dysfunction, whereas ISMN had no effect. In the three groups receiving a cholesterol diet, an increased extent of aortic lesions significantly correlated with increased endothelial dysfunction measured in the same preparations. The long-term treatment with PETN did not affect the vasorelaxing potency of PETN in aortic rings, and similar results were obtained in the case of ISMN. We conclude that long-term treatment with doses of PETN, which do not promote the development of in vitro vascular nitrate tolerance, may protect against atherosclerosis and endothelial dysfunction. This novel, yet unknown pharmacodynamic quality of nitrovasodilators like PETN may contribute to their long-term efficacy in coronary artery disease but may also imply new therapeutic indications in the future. |
| In vivo formation of 25-hydroxycholesterol from endogenous cholesterol after a single meal, dietary cholesterol challenge Johnson, K. A., C. J. Morrow, et al. (1994), J Lipid Res 35(12): 2241-53. Abstract: The role of oxysterols as regulatory molecules in the suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated in the intact rat in response to an acute dietary cholesterol challenge. When rats were fed highly purified cholesterol as a single meal at a level of 5% of the diet, maximal inhibition of enzyme activity (66%) occurred 120 min after the completion of the meal. Furthermore, when nonsaponifiable liver extracts were chromatographically resolved and analyzed by high performance liquid chromatography (HPLC) and capillary gas chromatography-mass spectrometry (GC-MS), 25-hydroxycholesterol was identified in the livers of rats 120 min after the completion of the single cholesterol meal. Significantly, only barely detectable amounts of 25-hydroxycholesterol were observed in the livers from control rats fed a sterol-free diet. The biosynthetic origin of 25-hydroxycholesterol was investigated with the use of deuterated water. Rats were fed deuterium oxide (33%) ad libitum for 3 days and then killed 120 min after the completion of a single cholesterol meal. As before, 25-hydroxycholesterol was detected in the livers from cholesterol-fed rats, but not to a significant extent in livers from control-fed rats receiving a sterol-free diet. Isotope ratio mass spectrometry revealed that the fractional incorporation of deuterium into 25-hydroxycholesterol (21%) was less than that observed for cholesterol (24%) isolated from the same livers, indicating that 25-hydroxycholesterol was produced endogenously from exogenous cholesterol and not from autoxidation of cholesterol. In a separate experiment it was also shown that 3Hmevalonate was incorporated into 25-hydroxycholesterol after a single meal cholesterol challenge, but was barely detected in the livers of control rats. The evidence obtained in the present article supports the hypothesis that 25-hydroxycholesterol is endogenously produced from cholesterol at early time intervals after an acute dietary cholesterol challenge. In addition, rat liver HMG-CoA reductase was inhibited by the administration of a single intragastric dose (1 microgram/kg) of an aqueous solution of 25-hydroxycholesterol. Thus, the results provide strong support for the conclusion that 25-hydroxycholesterol plays a significant role in the in vivo regulation of rat liver cholesterol biosynthesis after an acute dietary cholesterol challenge. |
| In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-Fed rabbits Channon, K. M., H. Qian, et al. (1998), Circulation 98(18): 1905-11. Abstract: BACKGROUND: The vascular endothelium is anatomically intact but functionally abnormal in preatherosclerotic states, and an early deficit in the bioavailability of nitric oxide (NO) or related molecules has been described in both humans and animal models. We hypothesized that the targeted gene transfer of NO synthase (NOS) isoforms might ameliorate or reverse the deficit. METHODS AND RESULTS: We constructed a recombinant adenovirus, Ad.nNOS, that expresses the neuronal isoform of NOS (nNOS) and used it for in vivo endovascular gene transfer to carotid arteries (CA) from normal and cholesterol-fed rabbits. Vessels were harvested 3 days after gene transfer. In CA from normal rabbits, Ad.nNOS generated high levels of functional nNOS protein predominantly in endothelial cells and increased vascular NOS activity by 3.4-fold relative to sham-infected control CA. Ad.nNOS gene transfer also significantly enhanced endothelium-dependent vascular relaxation to acetylcholine; at 3 micromol/L acetylcholine, Ad.nNOS-treated arteries showed an 86+/-4% reduction in precontracted tension, whereas control CA showed a 47+/-6% reduction in tension. Contraction in response to phenylephrine and relaxation in response to nitroprusside were unaffected in both control and Ad.nNOS-treated CA. To determine the effect of Ad.nNOS in atherosclerotic arteries, 10 male New Zealand White rabbits maintained on a 1% cholesterol diet for 10 to 12 weeks underwent gene transfer according to the same protocol used in normal rabbits. Ad.nNOS-treated arteries showed a 2-fold increase in NADPH-diaphorase staining intensity relative to sham-infected and Ad. betaGal-treated arteries. The CA from cholesterol-fed rabbits showed impaired acetylcholine-induced relaxation, but this abnormality was almost entirely corrected by Ad.nNOS gene transfer. CONCLUSIONS: In vivo adenovirus-mediated endovascular delivery of nNOS markedly enhances vascular NOS activity and can favorably influence endothelial physiology in the intact and atherosclerotic vessel wall. |
| In vivo implants of beta-sitosterol cause reductions of reactive cholesterol pools in mitochondria isolated from gonads of male goldfish (Carassius auratus) Leusch, F. D. and D. L. MacLatchy (2003), Gen Comp Endocrinol 134(3): 255-63. Abstract: beta-Sitosterol, a phytosterol found in high concentrations in pulp mill effluents, has been proposed as one of the causative agents for steroid depressions observed in fish exposed to pulp mill effluents. Previous studies have suggested a cholesterol-mediated mechanism; however, it is unknown how beta-sitosterol depresses gonadal steroidogenesis. In this study, adult male goldfish (Carassius auratus) were exposed for 24-31 days to beta-sitosterol (55% of a phytosterol mixture or 96% pure; 150 microg/g; Silastic implant) after which gonadal mitochondria were isolated. Pregnenolone production, an indicator of the size of the pool of reactive cholesterol, was then measured in the isolated mitochondria. Sterol exposure did not affect P450 side-chain cleavage enzyme (converts cholesterol to pregnenolone) activity but did decrease the size of the mitochondrial pool of reactive cholesterol, suggesting beta-sitosterol is impeding cholesterol transfer across the mitochondrial membrane. This finding is supported by the observation that 25-hydroxycholesterol, which passes through mitochondrial membranes without need for a membrane transporter, restores beta-sitosterol-induced reductions in pregnenolone production. |
| In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis Lee, W. N., S. Bassilian, et al. (1994), Am J Physiol 266(5 Pt 1): E699-708. Abstract: The synthesis of palmitate, stearate, and cholesterol in liver and nervous tissues (brain, cord, and nerve) of Sprague-Dawley rats was determined using deuterated water (D2O) and mass isotopomer analysis. Rats were given 4% deuterium in their drinking water after each receiving an intraperitoneal priming dose. Animals were killed at 1, 2, 4, and 8 wk for deuterium enrichment in body water and determination of mass isotopomer distribution in lipids from various tissues. In 1 wk, the enrichment in the body water reached a plateau of 2.6%, which is 65% of that in the drinking water. We observed the maximum incorporation number (N) in all lipids to be higher than those previously observed, being 22, 24, and 30 for liver palmitate, stearate, and cholesterol, respectively, and N may vary among tissues. Using a single exponential model, we found the half-time (t1/2) and the plateau levels of the newly synthesized lipids of the nervous tissues (t1/2 values ranging from 5 to 28 days) to be different from those of the liver (t1/2 values < or = 4 days) in this relatively long-term study. Mass isotopomer distribution analysis and D2O can be used not only to quantitate the replacement rate of many lipids in various compartments but may also be used to elucidate the tissue-specific synthetic pathways from N. |
| In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated Diraison, F., C. Pachiaudi, et al. (1996), Metabolism 45(7): 817-21. Abstract: Fractional lipid synthesis can be measured using the incorporation of deuterium from deuterated water. The calculations require knowledge of the maximum incorporation number (N) of deuterium atoms in the molecules synthesized. For both tissue palmitate and cholesterol, N values have been found to be higher during in vivo versus in vitro experiments. We determined the N values to be used for measuring the fractional synthesis of plasma cholesterol and of palmitate triglycerides (TG). Rats were given drinking water enriched (7% to 10%) with deuterated water, and N was determined from the mass isotopomer distributions of plasma cholesterol and plasma TG palmitate and the deuterium enrichment of plasma water. We found N to be 21 for palmitate and 27 for cholesterol. These values agree with those reported for tissue palmitate and cholesterol in vivo, and are higher than values found in vitro. We also found large deuterium enrichments in plasma glucose and in liver lactate and pyruvate. We suggest that, compared with in vitro studies, in vivo metabolism of these compounds leads to an additional pathway of incorporation of deuterium into lipids through deuterium-labeled acetyl coenzyme A (CoA). This could explain why N values are higher in vivo than in vitro. |