Inhibition of cholesterol synthesis by β-benzal butyric acid and derivatives

Cholesterol biosynthesis has been examined using rat liver slices in vitro from 2-¹⁴C-acetate and 2-¹⁴C-mevalonate, in the presence of β-benzal butyric acid (BBA) and its metabolite, α-hydroxy β-benzal butyric acid (HBBA), both of which are postulated to act as potential hypocholesterolemic agents. Procedures have been devised to follow radioactivity incorporation of these precursors into the squalene, lanosterol and cholesterol fractions. The results show that cholesterol synthesis from labeled acetate is noticeably inhibited by BBA final concentrations as small as 10 μM, while the rate of labeling is much less inhibited by HBBA. When acetate is replaced by labeled mevalonate, cholesterol synthesis is hardly inhibited by both BBA and HBBA. The results indicate that BBA probably affects some of the reactions which lead acetate to mevalonate formation. Acetyl-CoA: ligase (E.C.6.2.1.1) and acetyl-CoA acetyl transferase (E.C.2.3.1.) therefore have been examined. Ligase activity is substantially inhibited only by 1 mM concentration of BBA and HBBA, whereas the transferase enzyme is unaffected. BBA probably affects other reactions in the metabolic sequence which converts acetate into mevalonate. Presented in part at the International Symposium on Problems and Prospectives in Medical Therapy of Atherosclerosis, Parma, November, 1966.     

Inhibition of cholesterol synthesis by oxygenated sterols

Sterols derived from cholesterol by introducing a ketone or hydroxyl function in the 6, 7, 15, 20, 22, 24, or 25 positions are known to be potent inhibitors of sterol synthesis in cell cultures. To gain more information regarding structural requirements for inhibitory activity, inhibitory potencies were determined for a series of 18 C₂₇-steroids with various combinations of ketone and hydroxyl functions substituted in positions 3, 4, 5, 6, and 7, or with a single ketone or hydroxyl function in one of these positions. The effects of nuclear double bonds upon inhibitory activity were also examined. A ketone or hydroxyl function in position 3 and a second ketone or hydroxyl function in position 6 or 7 was required for inhibitory activity with two kinds of cell culture. A 3β5α6β-triol was not more inhibitory than a comparable 3β,6β-diol. Cholestance-3β 5α-diol inhibited sterol synthesis in L cells but not in liver cell cultures. The inhibitory activities of 7-oxygenated sterols were not markedly affected by the presence of a double bond in position 4 or 5. Current knowledge of the mechanism through which the oxygenated sterols suppress cholesterol synthesis is reviewed.     

Inhibition of cholesterol synthesis by lovastatin tested on six human cell typesin vitro

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 human lecithin cholesterol acyltransferase by monoterpenes

The lecithin-cholesterol acyltransferase activity of human plasma was found to be inhibited by Rowachol, a proprietary mixture of pure monoterpenes. Menthol, the major ingredient in Rowachol (32%), and a number of other monoterpenes were found to inhibit the enzyme independently. Concentrations of monoterpenes required to achieve 50% inhibition were of the same order of magnitude as the cholesterol concentration present in the reaction mixture.     

Regulation of cholesterol synthesis in isolated epithelial cells of human small intestine

We have investigated the regulation of cholesterol synthesis in isolated human small intestine epithelial cells (enterocytes). It was established that the amount of cholesterol synthesized increased linearly with the incubation time and the number of cells in the incubation mixture; the synthesis was suppressed by 7-ketocholesterol. Cholic, dehydrocholic, chenodeoxycholic, glycocholic, taurocholic, taurochenodeoxycholic and taurodeoxycholic acids inhibited cholesterol synthesis in enterocytes to different degrees in a dose-dependent manner. Lithocholic acid enhanced the rate of cholesterol synthesis. Deoxycholic acid, methyl ester of cholic acid and cholesterol did not affect the process. No bile acids tested, with the exception of taurodeoxycholic acid, affected fatty acid synthesis in enterocytes. Most bile acids also decreased cholesterol synthesis in cultured human skin fibroblasts. The results obtained make it possible to postulate that cholesterol synthesis in human enterocytes may be subject to a complex regulation by bile acids.     

Hepatic cholesterol synthesis from mevalonate and squalene in rats: Effect of feeding cholesterol supplemented diet during weaning and following starvation

The conversion of squalene to digitonin precipitable sterols by microsomes and soluble fractions from liver and the incorporation of mevalonate into nonsaponifiable lipids and digitonin precipitable sterols by 5000 g liver supernatant was studied in suckling rats and rats weaned on diet (Purina chow) supplemented with varying levels of cholesterol. The results indicate that the conversion of squalene to digitonin precipitable sterols was low in suckling rats and high in rats weaned on Purina chow diet. Weaning rats on 1% cholesterol supplemented diet effectively eliminated the post-weaning increase in mevalonate incorporation into nonsaponifiable and digitonin precipitable sterols, and in the conversion of squalene to digitonin precipitable sterols. The incorporation of mevalonate into nonsaponifiable lipids and digitonin precipitable sterols, and of squalene into digitonin precipitable sterols also was studied in liver preparations from adult rats which were starved and refed control or cholesterol supplemented diet. It was observed that the conversion of squalene to digitonin precipitable sterols by liver fractions from fasted rats was low, while that by liver fractions from rats refed control diet was higher. Furthermore, the post-fasting increase in the conversion of mevalonate to nonsaponifiable lipids and digitonin precipitable sterols and in squalene to digitonin precipitable sterols conversion essentially was eliminated by refeeding a 1% cholesterol supplemented diet. The low conversion of squalene to digitonin precipitable sterols in suckling rats, rats weaned on cholesterol supplemented diet, and adult rats that were starved, or starved and refed cholesterol supplemented diet, was due to the reduced activity of microsomal enzymes. It is concluded from this study that dietary cholesterol prevents the increase in cholesterol synthesis observed in developing and regenerating liver by suppressing the activities of one or more enzymes between mevalonate and squalene and between squalene and cholesterol.     

Inhibition of rat hepatic sterol formation from squalene by plasma lipoproteins

The conversion of³H-squalene to sterols by rat liver microsomes and cytosol was inhibited by individual rat and human plasma lipoproteins at various concentrations. This inhibition was also observed with added human high density apolipoprotein, but triglycerides, cholesterol or cholesteryl esters had no inhibitory effects. Lipoproteins and apo high density lipoprotein (HDL) were demonstrated to bind³H-squalene in vitro. The binding of³H-squalene by apo HDL could be reversed by increasing concentration of liver cytosol containing sterol carrier protein.     

Inhibition of ferrous-induced lipid peroxidation by pyrimido-pyrimidine derivatives in human liver membranes

The effects of pyrimido-pyrimidine derivatives (dipyridamole, RA-642, and RA-233) on lipid peroxidation, using d-α-tocopherol as standard, were studied in enriched membrane fractions from human and rat hepatocytes. Equimolar concentrations of ferrous sulfate and ascorbic acid were used to induce lipid peroxidation. The amount of peroxidized lipids observed in membrane fractions from human liver was smaller than in those from rat liver. In both species, however, pyrimido-pyrimidine derivatives, except for RA-233 in rat liver, inhibited lipid peroxidation dose-dependently in the following sequence: RA-642 > dipyridamole > d-α-tocopherol RA-233.     

The role of squalene synthetase in the inhibition of tetrahymanol biosynthesis by cholesterol inTetrahymena pyriformis

The biosynthesis of the triterpenoid alcohol tetrahymanol byTetrahymena pyriformis is rapidly inhibited by the addition of cholesterol to the growth medium. The primary site of this inhibition by cholesterol has been established to be at the level of the enzyme squalene synthetase. The protein synthesis inhibitor cycloheximide produces an identical decline in squalene synthetase activity to that of cholesterol and the half-life of the enzyme is about 50 minutes. No direct inhibition of the enzyme is observed and suggests that cholesterol inhibits the actual synthesis of the enzyme squalene synthetase. Farnesol is accumulated during in vitro incubations derived from cells grown in the presence of cholesterol or cycloheximide.     

Inhibition of hepatic cholesterol biosynthesis by chloroquine

Chloroquine is shown to be a potent inhibitor of cholesterol biosynthesis by isolated rat hepatocytes. Half-maximal inhibition of cholesterogenesis occurs at ca. 10μM chloroquine. Chloroquine does not affect fatty acid synthesis by isolated hepatocytes. This suggests that chloroquine acts on the cholesterol biosynthetic pathway beyond the cytosolic acetyl-CoA branchpoint of cholesterol and fatty acid synthesis.     

Stimulation of hepatic squalene and triglyceride synthesis by dimethylsulfoxide,in vitro

The incorporation of [¹⁴C] mevalonate and [¹⁴C] acetate into squalene by rat liver slices was increased over 7-fold by the presence of 5% dimethylsulfoxide (DMSO) in the incubation medium. The stimulation of squalene synthesis was dose-related over the concentration range of 1–5% DMSO and did not affect the incorporation of [¹⁴C] mevalonate, into the C₂₇-sterol fraction (cholesterol) but did increase (about 50%) incorporation into C₃₀-sterol (lanosterol) at a level, of 5% DMSO. The stimulation of squalene synthesis was observed under both anaerobic (N₂ atmosphere) and aerobic (ambient air or 95% O₂/5% CO₂) conditions and may represent a direct effect of DMSO on squalene synthetase. At a level of 5%, DMSO also stimulated 7-fold the incorporation of [¹⁴C] acetate into triglycerides by liver slices; this occurred without changes in incorporation into the phospholipid or free fatty acid fractions. The disproportionate increase in lipid labeling from [¹⁴C] acetate suggests that the effects of DMSO are not simply a matter of increasing [¹⁴C] acetate entry into the tissue.     

Inhibition of cholesterol synthesis and hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in rats by simvastatin and pravastatin

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 [2-¹⁴C]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 in mammary tissue,lung, and kidney following cholesterol feeding in the lactating rat

Pregnant rats were randomly allocated to one of 3 experimental dietary groups: Group 1–15.5% butter, 2% cholesterol, 0.78% sodium cholate purified diet; Group 2-standard rat diet with the addition of 10% lard and 2% cholesterol, and Group 3-standard rat diet. Plasma and milk cholesterol at 10 days postpartum were significantly elevated in dams fed exogenous cholesterol. The rat of incorporation of [1-¹⁴C] acetate into digitonin-precipitable sterols of mammary tissue slices from dams in Group 1 and Group 2 was eight-fold and two-fold, respectively, less than controls. Mammary tissue cholesterol was greater in dams fed dietary cholesterol. Thus, our data, for the first time, demonstrate that cholesterol synthesis in lactating rat mammary tissue is suppressed following cholesterol feeding. In a second experiment, the rate of incorporation of [1-¹⁴C] acetate into digitonin-precipitable sterols in kidney and lung tissue of Group 1 rats was suppressed; however, this response was not as marked as that observed in lactating mammary tissue. The concentration of cholesterol in kidney and lung was greater than controls. These results suggest that extrahepatic inhibition of cholesterol synthesis exists in the rat with a concomitant increase in tissue cholesterol.     

Inhibition of cholesterol biosynthesis by organosulfur compounds derived from garlic

The study was undertaken to test the inhibitory potential on cholesterogenesis of organosulfur compounds derived from garlic. The primary rat hepatocytes maintained in Dulbecco's modified Eagle's medium were treated with [2-¹⁴C]-acetate as substrate for cholesterol synthesis in the presence or absence of test compounds at 0.05 to 4.0 mmol/L. Eleven watersoluble and six lipid-soluble compounds of garlic were tested. Among water-soluble compounds,S-allyl cysteine (SAC),S-ethyl cysteine (SEC), andS-propyl cysteine (SPC) inhibited [2-¹⁴C]acetate incorporation into cholesterol in a concentration-dependnet manner, achieving 42 to 55% maximal inhibition. γ-Glutamyl-S-allyl cysteine, γ-glutamyl-S-methyl cysteine, and γ-glutamyl-S-propyl cysteine were less potent, exerting only 16 to 29% maximal inhibitions. Alliin,S-allyl-N-acetyl cysteine,S-allylsulfonyl alanine, andS-methyl cysteine had no effect on cholesterol synthesis. Of the lipid-soluble compounds, diallyl disulfide (DADS), diallyl trisulfide (DATS), and dipropyl disulfide (DPDS) depressed cholesterol synthesis by 10 to 25% at low concentrations (0.5 mmol/L), and abolished the synthesis at high concentrations (1.0 mmol/L). Diallyl sulfide, dipropyl sulfide, and methyl allyl sulfide slightly inhibited [2-¹⁴C]acetate incorporation into cholesterol only at high concentrations. The complete depression of cholesterol synthesis by DADS, DATS, and DPDS was associated with cytotoxicity as indicated by marked increase in cellular LDH release. There was no apparent increase in LDH secretion by water-soluble compounds exceptS-allyl mercaptocysteine, which also abolished cholesterol synthesis. Judging from maximal inhibition and IC₅₀ (concentration required for 50% of maximal inhibition), SAC, SEC, and SPC are equally potent in inhibiting cholesterol synthesis.     

Inhibition by 5-(tetradecyloxy)-2-furoic acid of fatty acid and cholesterol synthesis in isolated rat hepatocytes

Fatty acid and cholesterol synthesis in isolated rat hepatocytes were strongly inhibited by 5-(tetradecyloxy)-2-furoic acid. With either³H₂O or [2-¹⁴C]acetate as the labeled precursor, the concentrations of inhibitor causing 50% decrease in fatty acid and cholesterol synthesis were, respectively, <0.005 mM and 0.020 mM. At 0.1 mM inhibitor, citrate concentration in cells from fed rats was increased by 75%; lactate and pyruvate concentrations were decreased by 30%; ethanol oxidation was decreased by 20%; with cells from starved rats, the mitochondrial [NAD⁺]/[NADH] was decreased. Other parameters were unaffected. Both its potency and its specificity indicate that 5-(tetradecyloxy)-2-furoic acid will be useful in studies on the regulation of lipid biosynthesis.     

Effect of alimentation on human serum squalene levels

Five subjects consumed a meal containing ca. 1.5 mg squalene. Postprandial serum samples showed that in three of the subjects squalene and lipid levels reached maxima at 3 hr. Squalene levels in the low density lipoproteins of these subjects also increased. The results indicate that alimentation is a major factor in the variation in serum squalene levels.     

对甲酚衍生物催化氧化合成对羟基苯甲醛衍生物

阐述了对甲酚衍生物催化氧化合成对式苯甲醛衍生物的研究状况及近期研究动态,对Ｃｏ盐催化剂、Ｃｏ／Ｃｕ双金属催化剂以及其他催化剂对对甲酚衍生物选择性催化氧化的性能作一评述。     

Inhibition of Mycobacterium tuberculosis methionine aminopeptidases by bengamide derivatives

Methionine aminopeptidase (MetAP) carries out an essential function of protein N‐terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X‐ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non‐replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.     

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-¹⁴C and mevalonate-5-³H into squalene, C₃₀ sterols, and C₂₇ sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of³H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas¹⁴C-acetate conversion was reduced to ca. 35% of control values during the same period.³H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as¹⁴C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from¹⁴C-acetate and³H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.