Cholesterol Journal Articles

Record 7661 to 7680

Inhibition of cholesterol synthesis and hepatic 3-hydroxy-3-methylglutaryl--CoA reductase in rats by simvastatin and pravastatin
Del Puppo, M., S. Rauli, et al. (1995), Lipids 30(11): 1057-61.
Abstract: In this communication we attempt to provide one possible explanation for the observed differences regarding kinetics and distribution between simvastatin and pravastatin. Rats treated with simvastatin or pravastatin exhibited a reduction in the incorporation of 2-(14)C acetate into liver cholesterol and displayed lower plasma mevalonate levels as compared to control animals. Moreover, both the total and dephosphorylated 3-hydroxy-3-methylglutaryl--CoA (HMG-CoA) reductase (EC 1.1.1.34) activities, particularly 1 h after treatment, were greatly reduced in liver microsomes obtained from simvastatin-treated as compared to control rats. During the same time frame, these parameters were actually elevated with pravastatin treatment. It is known that HMG-CoA reductase synthesis and activity increase following their competitive inhibition. Our results suggest that pravastatin, at 1 h following treatment, was no longer bound to the enzyme; however, it had entered the liver because its inhibitory effect on cholesterol synthesis was manifest at early times after administration. These data provide a plausible rationale for the earlier observation that activity of simvastatin persists longer in plasma than does that of pravastatin.

Inhibition of cholesterol synthesis by atorvastatin in homozygous familial hypercholesterolaemia
Raal, F. J., A. S. Pappu, et al. (2000), Atherosclerosis 150(2): 421-8.
Abstract: Patients with homozygous familial hypercholesterolaemia (HoFH) have markedly elevated low density lipoprotein (LDL) cholesterol levels that are refractory to standard doses of lipid-lowering drug therapy. In the present study we evaluated the effect of atorvastatin on steady state concentrations of plasma lipids and mevalonic acid (MVA), as well as on 24-h urinary excretion of MVA in patients with well characterized HoFH. Thirty-five HoFH patients (18 males; 17 females) received 40 mg and then 80 mg atorvastatin/day. The dose of atorvastatin was increased further to 120 mg/day in 20 subjects and to 160 mg/day in 13 subjects who had not achieved LDL cholesterol goal, or in whom the dose of atorvastatin had not exceeded 2.5 mg/kg body wt per day. LDL cholesterol levels were reduced by 17% at the 40 mg/day and by 28% at the 80 mg/day dosage (P<0.01). Reduction in LDL cholesterol in the five receptor negative patients was similar to that achieved in the 30 patients with residual LDL receptor activity. Plasma MVA and 24-h urinary excretion of MVA, as markers of in vivo cholesterol synthesis, were elevated at baseline and decreased markedly with treatment. Urinary MVA excretion decreased by 57% at the 40 mg/day dose and by 63% at the 80 mg/day dosage (P<0. 01). There was a correlation between reduction in LDL cholesterol and reduction in urinary MVA excretion; those patients with the highest basal levels of MVA excretion and thus the highest rates of cholesterol synthesis having the greatest reduction in LDL cholesterol (r=0.38; P=0.02). Increasing the dose of atorvastatin to 120 and 160 mg/day did not result in any further reduction in LDL cholesterol or urinary MVA excretion suggesting a plateau effect with no further inhibition of cholesterol synthesis at doses of atorvastatin greater than 80 mg/day.

Inhibition of cholesterol synthesis by cyclopropylamine derivatives of squalene in human hepatoblastoma cells in culture
Van Sickle, W. A., M. R. Angelastro, et al. (1992), Lipids 27(3): 157-60.
Abstract: Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualene N-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol synthesis in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited 3H-squalene conversion to 3Hsqualene epoxide in microsomes (IC50 = 5.0 microM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualene N-methylcyclopropylamine inhibited 3Hsqualene conversion to 3Hlanosterol (IC50 = 12.0 microM) and caused 3H-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from 14Cacetate in HepG2 cells was inhibited by both derivatives (IC50 = 1.0 microM for trisnorsqualene cyclopropylamine; IC50 = 0.5 microM for trisnorsqualene N-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated 14Csqualene, while cells incubated with trisnorsqualene N-methylcyclopropylamine accumulated 14Csqualene epoxide and 14Csqualene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.

Inhibition of cholesterol synthesis by lovastatin tested on six human cell types in vitro
Sviridov, D. D., I. G. Safonova, et al. (1990), Lipids 25(3): 177-9.
Abstract: Lovastatin (mevinolin) caused a strong and dose-dependent inhibition of cholesterol synthesis in six types of cultured human cells. Fifty percent inhibition of cholesterol synthesis in human enterocytes was observed at a lovastatin concentration of about 0.004 ng/ml and in other cells at a lovastatin concentration of about 0.03 ng/ml. At lovastatin concentrations between 1.0 and 100.0 ng/ml, a moderate tissue selectivity of lovastatin action was noted. At optimal concentrations, lovastatin inhibited cholesterol synthesis in hepatocytes by 98%, in normal and LDL-receptor negative fibroblasts, arterial smooth muscle cells and hepatoma G-2 cells by about 90%, and in enterocytes by 75%. In rat enterocytes lovastatin inhibited cholesterol synthesis by only 60%.

Inhibition of cholesterol synthesis by squalene synthase inhibitors does not induce myotoxicity in vitro
Flint, O. P., B. A. Masters, et al. (1997), Toxicol Appl Pharmacol 145(1): 91-8.
Abstract: The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previous in vitro study, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-induced in vitro myotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis (3Hleucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis (14Cacetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-induced in vitro myotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that depletion of metabolites of geranylgeranyl pyrophosphate, and not inhibition of cholesterol synthesis, is the primary cause of HMG CoA reductase-induced myotoxicity.

Inhibition of cholesterol synthesis causes both hypercholesterolemia and hypocholesterolemia in hamsters
Sawada, M., M. Matsuo, et al. (2002), Biol Pharm Bull 25(12): 1577-82.
Abstract: Effects of FR194738 ((E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-2-methyl-2-(3-thienylmeth oxy)propyloxybenzylamine hydrochloride), a squalene epoxidase inhibitor, on lipid metabolism were compared with those of pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in hamsters. Drugs were given for 10 d either as a diet mixture or as a bolus oral gavage, and similar results were obtained with each type of administration. FR194738 (0.01-0.32% as a diet mixture; 10-100 mg/kg as an oral gavage) dose-dependently decreased serum total cholesterol, non high density lipoprotein (HDL) cholesterol, HDL cholesterol and triglyceride levels, and the changes in serum parameters were similar. Pravastatin (0.01-0.32% as a diet mixture; 1-100 mg/kg as an oral gavage) increased serum cholesterol levels, and dose-dependently decreased serum triglyceride levels. Although oral gavage of FR194738 at 32 mg/kg and pravastatin at 3.2 and 10 mg/kg increased hepatic HMG-CoA reductase activity, the degree of the changes was far greater with the latter than the former drug. FR194738 slightly increased hepatic cholesterol content at 32 mg/kg, whereas pravastatin dose-dependently increased hepatic cholesterol content until it leveled off at 32 and 100 mg/kg. It is concluded that inhibition of squalene epoxidase and HMG-CoA reductase triggers both hypercholesterolemic (hepatic cholesterol synthesis) and hypocholesterolemic (hepatic cholesterol uptake) mechanisms. FR194738 appears to induce a greater enhancement of the latter rather than the former, whereas pravastatin has a greater effect on the former.

Inhibition of cholesterol synthesis ex vivo and in vivo by fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase
Yamamoto, A., S. Itoh, et al. (1995), Experientia 51(3): 223-6.
Abstract: The inhibitory effect of fluvastatin sodium (fluvastatin), a new type of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A inhibitor, on de novo cholesterol synthesis was investigated and compared with that of pravastatin. Fluvastatin at a concentration of 12.5 mg/kg inhibited sterol synthesis ex vivo from 14Cacetate in rat liver and ileum by 97-99% with respect to the control, while the inhibition in kidney was 55%. The inhibition by fluvastatin in the liver and ileum persisted for approximately 9 h after administration. Significant differences between fluvastatin also had an inhibitory effect on cholesterol synthesis in vivo in various tissues of rats given 14Cacetate intraperitoneally. Sterol synthesis in the liver, ileum and kidney was inhibited by over 95% 3 h after administration of 6.25 mg/kg of fluvastatin. Significant differences between fluvastatin and pravastatin were found in the liver and ileum. Fluvastatin was more potent than pravastatin in inhibiting both ex vivo and in vivo sterol synthesis in the ileum (but not in kidney) and liver.

Inhibition of cholesterol synthesis from mevalonate by aminotriazole treatment in vivo
Hashimoto, F., C. Sugimoto, et al. (1990), Chem Pharm Bull (Tokyo) 38(9): 2532-6.
Abstract: 3-Amino-1,2,4-triazole (aminotriazole) is an irreversible inhibitor of catalase which is a marker enzyme of peroxisomes. We studied the effect of aminotriazole treatment on biosyntheses of cholesterol and bile acid in vivo. When catalase activity of peroxisomes of rat liver was inhibited by aminotriazole treatment, bile acid content in the bile was significantly decreased to about 70% of the control, but that in the liver was not changed. Cholesterol content in the bile was significantly decreased to about 80% of the control, while in liver and serum the content was not significantly changed. When 2-14Cmevalonate was administered to rats, radioactivities of cholesterol in the liver, serum and bile were all drastically decreased by aminotriazole treatment, and an unidentified radioactive product was detected. Radioactivity of bile acid in the bile was also greatly decreased. In a similar experiment with 4-14Ccholesterol, aminotriazole treatment had no effect on the radioactivity of either cholesterol or bile acid in the liver, serum and bile. In this case, the unidentified product could not be detected. These results indicate that when catalase activity of liver peroxisomes is suppressed by aminotriazole treatment, biosynthesis of bile acid from exogenous cholesterol is not inhibited, but a step in the pathway of biosynthesis of endogenous cholesterol from mevalonate is inhibited.

Inhibition of cholesterol synthesis in vitro by extracts and isolated compounds prepared from garlic and wild garlic
Sendl, A., M. Schliack, et al. (1992), Atherosclerosis 94(1): 79-85.
Abstract: Using a modified liver homogenate model to assay for the inhibition of cholesterol biosynthesis, different garlic and wild garlic extracts as well as pure compounds isolated from them were investigated for their influence on cholesterol synthesis. Chloroform and acetone/chloroform extracts of garlic and wild garlic inhibited cholesterol synthesis 44-52% at a concentration of 166 micrograms/ml, while the 5 individual sulfur-containing compounds ajoene, methylajoene, allicin, 2-vinyl-4H-1,3-dithiin and diallydisulfide inhibited cholesterol synthesis by 37-72% (10(-3) M corresponding to 234, 208, 162, 144, 146 micrograms/ml, respectively). Ajoene, 2-vinyl-4H-1,3-dithiin and allicin show IC50 values of 6.4, 7.2 and 9.4 x 10(-4) M, respectively. The results demonstrate that garlic and wild garlic may reduce serum cholesterol levels primarily by inhibiting cholesterol synthesis if taken in sufficient amount and that this effect arises from a mixture of multiple compounds from the sulfur-containing class of thiosulfinates, ajoenes and dithiines. Wild garlic extracts showed nearly identical efficiency to garlic extracts.

Inhibition of cholesterylester accumulation by 17 beta-estradiol in macrophages through activation of neutral cholesterol esterase
Tomita, T., F. Sawamura, et al. (1996), Biochim Biophys Acta 1300(3): 210-8.
Abstract: Premenopausal women are at a lower risk of coronary heart disease relative to age matched men. However, the underlying mechanisms are not clearly understood. This article studies the effects of 17 beta-estradiol (17 beta-E2) at physiological concentrations on the cholesterylester metabolism in macrophages (J774 A.1 cells) with a particular focus on neutral cholesterol esterase (N-CEase). Cells were incubated with beta-VLDL, 1-14Coleic acid and 17 beta-E2 (0.25 and 2.5 nM). 17 beta-E2 dose-dependently reduced cholesteryl-1-14Coleate (14C-CO) at 36 h and 48 h relative to the control. It also stimulated hydrolysis of 14C-CO in foam cells on 36 h and 48 h incubation. In addition, 17 beta-E2 markedly increased N-CEase activity at 24 h and 36 h. This increase preceded the enhanced hydrolysis of cholesterylester, 17 alpha-E2 (inactive isomer), estrone and estriol had no stimulatory action on N-CEase, whereas progesterone and testosterone inhibited it. 17 beta-E2-treatment (24 h) increased the activity of cyclic AMP-dependent protein kinase (A-kinase). DEAE-cellulose column chromatography revealed that an isoform (type II) of A-kinase appeared in 17 beta-E2-treated cells in addition to type I of A-kinase found in the control cells. These results suggest that inhibition of cholesterylester accumulation in macrophages by 17 beta-E2 is mediated by an enhancement of N-CEase activity possibly through an increase in A-kinase.

Inhibition of cholinesterase activity by tetrahydroaminoacridine and the hemisuccinate esters of tocopherol and cholesterol
Chelliah, J., J. D. Smith, et al. (1994), Biochim Biophys Acta 1206(1): 17-26.
Abstract: The anticholinesterase properties of tetrahydroaminoacridine (THA, Tacrine), alpha-tocopheryl hemisuccinate (TS), and cholesteryl hemisuccinate (CS), given alone and in combination, were examined in vitro. Results from these studies indicate that: 1 THA is a potent inhibitor of acetylcholinesterase (AChE, IC50 of 0.40 microM) and butyrylcholinesterase (BChE, IC50 of 0.10 microM) with greatest inhibitory activity towards BChE; 2 TS and CS are weak inhibitors of BChE (IC50 of 100 microM and 168 microM, respectively) but potent inhibitors of ACHE (IC50 of 1.73 microM and 0.79 microM, respectively); 3 both TS and CS treatment in combination with THA significantly increased THA's anticholinesterase activity. The percentage AChE inhibition observed with this combination was often significantly greater than the sum of the individual values (synergistic). The addition of 0.5 microM CS or TS to an ACHE preparation reduced THA's IC50 value from 0.40 microM or 0.18 microM, respectively 4; inhibition of AChE by THA, TS and CS are mixed non-competitive while THA inhibition of BChE is mixed non-competitive and TS and CS inhibition of BChE are simple non-competitive; and 5 inhibition of cholinesterases by TS and CS occurs immediately (50 to 75%), during the first 30 min of incubation (25 to 50%) and is dependent on the anionic charged portion of the molecule. In conclusion, our experimental data indicate that TS and CS are potent inhibitors of AChE activity and significantly potentiate the anticholinesterase activity of THA. Such potent and synergistic inhibition of AChE suggest that TS or CS, alone and in combination with THA, may prove beneficial in the treatment of organophosphate poisoning and Alzheimer's disease.

Inhibition of dietary cholesterol ester absorption by 3-BCP, a suicide inhibitor of cholesterol-esterase
Bailey, J. M., L. L. Gallo, et al. (1995), Biochem Soc Trans 23(3): 408S.

Inhibition of endothelial lipase causes increased HDL cholesterol levels in vivo
Jin, W., J. S. Millar, et al. (2003), J Clin Invest 111(3): 357-62.
Abstract: Endothelial lipase (EL) is a recently discovered member of the lipoprotein lipase gene family that hydrolyzes HDL phospholipids ex vivo and reduces HDL cholesterol (HDL-C) levels when overexpressed in vivo in mice. To gain further insight into the physiological role of EL in the metabolism of HDL in vivo, studies were performed in which EL was inhibited in wild-type, hepatic lipase knockout (HL(-/-)), and human apoA-I transgenic mice by intravenous infusion of a polyclonal antibody inhibitory to murine EL. As compared with infusion of a control antibody, infusion of the inhibitory antibody resulted in a 25-60% increase in HDL-C levels in the three mouse models, with the peak HDL-C levels occurring at 48 hours after injection. Inhibition of EL also generated larger HDL particles in the HL(-/-) mice. The clearance of HDL phospholipid was significantly slower in human apoA-I transgenic mice injected with an antibody against murine EL (mEL) than in mice injected with a control antibody. We conclude that inhibition of EL results in increased HDL-C levels and that EL is an important enzyme in the physiological regulation of HDL metabolism.

Inhibition of expression of P-selectin by antioxidant in cholesterol-fed rats
Lee, C. S., J. M. Choi, et al. (1999), J Korean Med Sci 14(1): 8-14.
Abstract: Butylated hydroxytoluene (BHT) can inhibit experimental atherosclerosis in animals. Although the agent is an antioxidant, the exact mechanism of the reaction in atherosclerosis is still unknown. To investigate the effects of BHT on expression of P-selectin (PADGEM, GMP-140), intercellular adhesion molecule-1 (ICAM-1) and class II MHC (Ia) antigen, we proposed an experiment on rats. Male rats (n=18 per group) were fed either a normal cholesterol control diet, a normal cholesterol diet containing 0.5% BHT (BD), a high cholesterol diet containing 1.5% cholesterol and 0.1% sodium cholate (CD), or the CD diet containing 0.5% BHT (BCD). Rats were sacrificed after 3 days, and after 1, 2, 4, 10, and 17 weeks of dietary treatment. Although there was no gross or light microscopic atherosclerotic lesions, scanning electron microscopy revealed monocytic adhesion to aortic endothelium and mild endothelial injuries in CD and BCD groups. Immunohistochemically, the addition of BHT to a high cholesterol diet inhibited P-selectin expression but not in ICAM-1 and Ia antigen. These findings suggest that in rats, high cholesterol diets induce expression of ICAM-1, P-selectin and Ia antigen. In addition, the antiatherogenic effect of BHT may play a role in the inhibition of P-selectin.

Inhibition of expression of the multidrug resistance-associated P-glycoprotein of by phosphorothioate and 5' cholesterol-conjugated phosphorothioate antisense oligonucleotides
Alahari, S. K., N. M. Dean, et al. (1996), Mol Pharmacol 50(4): 808-19.
Abstract: Multiple drug resistance (MDR) as a result of overexpression of the P-glycoprotein drug transporter, a product of the MDR1 gene, is a significant problem in cancer therapeutics. We demonstrate that phosphorothioate antisense oligonucleotides can reduce levels of MDR1 message, inhibit expression of P-glyco protein, and affect drug uptake in MDR mouse 3T3 fibroblasts. An obligonucleotide (5995) directed against a sequence overlapping the AUG start codon was effective in reduction MDR1 transcript and protein levels when used at submicromolar concentrations in conjunction with cationic liposomes, whereas a scrambled control oligonucleotide (10221) was ineffective. Substantial and specific antisense effects could also be attained with a 5' cholesterol conjugate of the 5995 sequence. In this case, use of cationic liposomes was unnecessary. The 5' cholesterol 5995, but the not 5' cholesterol 10221, reduced MDR1 message and P-glycoprotein levels by 50-60% when used at low micromolar concentrations. In parallel, treatment with 5' cholesterol 5995 also enhanced cellular accumulation of rhodamine 123, a well-known substrate of the P-glycoprotein transporter. The effectiveness of the cholesterol-conjugated 5995 may be due to its rapid and extensive cell uptake, as indicated in flow cytometry and confocal microscopy studies. These observations suggest that cholesterol-conjugated anti-sense oligonucleotides may offer a novel approach to inhibition of P-glycoprotein-mediated MDR and to the modulation of other tumor cell genes whose overexpression contributes to the neoplastic state or to resistance to therapy.

Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase
Henin, N., M. F. Vincent, et al. (1995), Faseb J 9(7): 541-6.
Abstract: AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.

Inhibition of free radical-mediated cholesterol peroxidation by diazeniumdiolate-derived nitric oxide: effect of release rate on mechanism of action in a membrane system
Korytowski, W., M. Zareba, et al. (2000), Chem Res Toxicol 13(12): 1265-74.
Abstract: Nitric oxide ((*)NO) flux in relation to antiperoxidant action has been studied, using large unilamellar liposomes (LUVs) as target membranes. LUVs consisting of an oxidizable phosphatidylcholine (PC), (14)Ccholesterol (Ch) as a reaction probe, and 5alpha-hydroperoxycholesterol (5alpha-OOH) as a nonregenerable primer underwent chain peroxidation when exposed to a lipophilic iron chelate Fe(HQ)(3), 1 microM and ascorbate (AH(-), 1 mM) at 37 degrees C. Reaction progress was monitored by (i) HPLC with reductive-mode electrochemical detection to assess the decay of 5alpha-OOH and the formation and/or turnover of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH) and (ii) HPTLC with phosphorimaging to track all major (14)C-labeled oxidation products (ChOX), including 7alphabeta-OOH, 7alpha-OH, 7beta-OH, and 5,6-epoxide. Three diazeniumdiolate (*)NO donors with different half-lives were tested for their ability to interfere with peroxidation: MANO (approximately 1 min), PANO (15 min), and SPNO (38 min). At starting concentrations of < or =200 microM, none of the donors slowed 5alpha-OOH exponential decay, ruling out any interference with redox-active iron. However, SPNO and to a greater extent PANO (but not the decomposed donors) decreased both the initial rate and steady state of 7alphabeta-OOH accumulation in a strong dose-dependent fashion. In contrast, MANO completely inhibited 7alphabeta-OOH formation over the first 5 min of reaction, but thereafter, the peroxide accumulated rapidly, albeit more slowly than without MANO and independently of the MANO dose. The latter response diminished with increasing Fe(HQ)(3) concentration, coincident with more rapid 5alpha-OOH loss. The same general trends with MANO, PANO, and SPNO were observed when the entire population of (14)CChOX species was monitored. These effects are attributed to interception of Ch- and PC-derived free radicals by (*)NO, high-flux (*)NO from MANO acting mainly on 5alpha-OOH-derived radicals (chain prevention), low-flux (*)NO from SPNO mainly on downstream radicals (chain termination), and intermediate-flux (*)NO from PANO by a combination of these mechanisms. Thus, delivery rate can be an important determinant of how (*)NO inhibits peroxide-induced lipid peroxidation.

Inhibition of gallbladder emptying decreases cholesterol saturation in bile in the Richardson ground squirrel
Pauletzki, J. G., Q. W. Xu, et al. (1995), Hepatology 22(1): 325-31.
Abstract: Impaired gallbladder emptying is frequent in cholesterol gallstone disease as well as in predisposing conditions like pregnancy and obesity. Gallbladder hypomotility is considered a pathogenic factor for gallstone formation, providing the residence time for cholesterol crystal nucleation, but any effect on the enterohepatic circulation of bile acids and subsequently on biliary lipid composition is unknown. Therefore, we studied the effect of prolonged suppression of gallbladder emptying with a cholecystokinin (CCK-A) receptor antagonist on bile formation in Richardson ground squirrels fed a trace versus a 1% cholesterol diet. Biliary lipid secretion was measured directly and bile acid pool size assessed by isotope dilution (14C-cholic acid). Gallbladder contraction was determined in vitro in response to CCK. The CCK-antagonist (MK-329) greatly inhibited gallbladder contraction in vitro and increased gallbladder fasting volume and bile acid pool size in vivo. It significantly lowered the cholesterol saturation index by 35% and 46% in hepatic bile and by 18% and 28% in gallbladder bile in the trace and cholesterol diet groups, respectively. Bile acid secretion and bile flow doubled with the CCK-receptor antagonist. Chronic CCK receptor antagonist-induced inhibition of gallbladder emptying increases bile acid secretion and thereby decreases cholesterol saturation in bile. Extensive biliary hypomotility thus leads to a more rapid cycling of bile acids by depriving the gallbladder of its function in the enterohepatic circulation.

Inhibition of hepatic ACAT decreases ApoB secretion in miniature pigs fed a cholesterol-free diet
Huff, M. W., D. E. Telford, et al. (1994), Arterioscler Thromb 14(9): 1498-508.
Abstract: To test the hypothesis that hepatic cholesteryl ester is involved in the regulation of apolipoprotein (apo) B secretion into plasma, apoB kinetic studies were performed in six control miniature pigs and in six pigs after a 21-day administration of the acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor DuP 128 (2.2 mg.kg-1.d-1 i.v.). Pigs were fed low-fat, cholesterol-free diets. Total plasma cholesterol, triglyceride, very-low-density lipoprotein (VLDL) triglyceride, and low-density lipoprotein (LDL) cholesterol decreased 18%, 29%, 40%, and 26% respectively (P <.03). 131I-VLDL and 125I-LDL were injected simultaneously into each animal, and apoB kinetics were analyzed by using multi-compartmental analysis (SAAM30). VLDL apoB pool size decreased significantly by 60% (0.32 versus 0.84 mg/kg), which was due to a 65% reduction in the VLDL apoB production or secretion rate (1.03 versus 2.94 mg.kg-1.h-1). The fractional catabolic rate was unchanged. LDL apoB pool size decreased nonsignificantly by 18% (5.61 versus 6.90 mg/kg) due entirely to a 24% decrease in production rate (0.26 versus 0.34 mg.kg-1.h-1). At necropsy, hepatic microsomal ACAT activity decreased by 68% (0.28 versus 0.88 nmol.min-1.mg-1; P <.0002). Although an increase in hepatic free cholesterol leading to a decreased LDL receptor expression might be expected, this did not occur. The concentration of hepatic cholesterol and the LDL apoB fractional catabolic rate were unaffected by DuP 128. In addition, the concentration of hepatic triglyceride and the activity of diacylglycerol acyltransferase were not altered by DuP 128, indicating a lack of effect of DuP 128 on hepatic triglyceride metabolism. We conclude that inhibition of hepatic cholesteryl ester synthesis in vivo decreases apoB secretion into plasma.

Inhibition of hepatic cholesterol biosynthesis by a 3-hydroxy-3-methylglutaryl coenzyme A synthase inhibitor, 1233A, in mice
Nagashima, H., H. Kumagai, et al. (1993), Life Sci 52(19): 1595-600.
Abstract: We have studied the in vivo inhibition of hepatic sterol biosynthesis by 1233A, a specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase. Administration of the compound orally to mice resulted in a dose-dependent inhibition of 14Cacetate incorporation into sterols in liver, but did not exert any significant effect on 14Cmevalonate incorporation. The results indicate that the in vivo inhibition of sterol synthesis occurs only at pre-mevalonate enzymatic steps in the sterol biosynthetic pathway, thus being compatible with the presumed site of inhibition, HMG-CoA synthase. Moreover, owing to irreversible inactivation of the enzyme by 1233A, it was possible to detect in vivo effect on the enzyme by assays of its activity in cell-free extracts from livers; the drug-treatment also resulted in a similarly dose-dependent inhibition of HMG-CoA synthase activity. In contrast, acetoacetyl-CoA thiolase and HMG-CoA reductase, the enzymes also responsible for mevalonate synthesis in the pathway, did not show any significant change in activity. These results clearly demonstrate that the inhibition of hepatic sterol synthesis caused by 1233A is indeed due to selective inhibition of HMG-CoA synthase in the tissues.

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