Genome mining and biosynthesis of the Acyl-CoA:cholesterol acyltransferase inhibitor beauveriolide I and III in Cordyceps militaris.

Ascomycete fungi Cordyceps are widely used in traditional Chinese medicine, and numerous investigations have been carried out to uncover their biological activities. However, primary researches on the physiological effects of Cordyceps were committed using crude extracts. At present, there are only a few compounds which were comprehensively characterized from Cordyceps, partial owing to the low production. In order to scientifically take advantage of Cordyceps, we used the strategy of genome mining to discover bioactive compounds from Cordyceps militaris. We found the putative biosynthetic gene cluster of the acyl-CoA:cholesterol acyltransferase inhibitor beauveriolides in the genome of C. militaris, and produced the compounds by heterologous expression in Aspergillus nidulans. Production of beauveriolide I and III also was detected in both ferment mycelia and fruiting bodies of C. militaris. The possible biosynthetic pathway was proposed. Our studies unveil the active compounds of C. militaris against atherosclerosis and Alzheimer's disease and provide the enzyme resources for the biosynthesis of new cyclodepsipeptide molecules.

Effect of corosolic acid on dietary hypercholesterolemia and hepatic steatosis in KK-Ay diabetic mice.

Corosolic acid (CA), contained in the leaves of the banaba plant (Lagerstroemia speciosa L.), is a pentacyclic triterpene, and has hypoglycemic effects. The effects of CA on dietary hypercholesterolemia and hepatic steatosis were assessed in KK-Ay mice, an animal model of type 2 diabetes. Two kinds of high cholesterol diet with or without 0.023% CA, were prepared for the study. KK-Ay mice were fed a normal diet (controls), the high cholesterol diet with CA (CA-mice) or that without CA (HC-mice) for 10 weeks. CA inhibited the mean blood cholesterol level by 32% (P<0.05) and the liver cholesterol content by 46% (P<0.05) compared with those of HC-mice 10 weeks after the start of dietary intake. Acutely, CA inhibited the mean blood cholesterol level 4 h after the administration of a high-cholesterol cocktail in an oral cholesterol-loading test, compared with that of control mice (P<0.05). These results suggest that CA has some direct effects on the cholesterol absorption process in the small intestine. CA may inhibit the activity of cholesterol acyltransferase, which acts in the re-esterification of cholesterol in the small intestine, in type 2 diabetes.

Esculeogenin A, a new tomato sapogenol, ameliorates hyperlipidemia and atherosclerosis in ApoE-deficient mice by inhibiting ACAT.

OBJECTIVE: We recently identified esculeoside A, a new spirosolane-type glycoside, with a content in tomatoes that is 4-fold higher than that of lycopene. In the present study, we examined the effects of esculeoside A and esculeogenin A, a new aglycon of esculeoside A, on foam cell formation in vitro and atherogenesis in apoE-deficient mice. METHODS AND RESULTS: Esculeogenin A significantly inhibited the accumulation of cholesterol ester (CE) induced by acetylated low density lipoprotein (acetyl-LDL) in human monocyte-derived macrophages (HMDM) in a dose-dependent manner without inhibiting triglyceride accumulation, however, it did not inhibit the association of acetyl-LDL to the cells. Esculeogenin A also inhibited CE formation in Chinese hamster ovary cells overexpressing acyl-coenzymeA (CoA): cholesterol acyl-transferase (ACAT)-1 or ACAT-2, suggesting that esculeogenin A suppresses the activity of both ACAT-1 and ACAT-2. Furthermore, esculeogenin A prevented the expression of ACAT-1 protein, whereas that of SR-A and SR-BI was not suppressed. Oral administration of esculeoside A to apoE-deficient mice significantly reduced the levels of serum cholesterol, triglycerides, LDL-cholesterol, and the areas of atherosclerotic lesions without any detectable side effects. CONCLUSIONS: Our study provides the first evidence that purified esculeogenin A significantly suppresses the activity of ACAT protein and leads to reduction of atherogenesis.

Effects of trans-10,cis-12 conjugated linoleic acid on cholesterol metabolism in hypercholesterolaemic hamsters.

BACKGROUND: Conjugated linoleic acid (CLA) has received great attention in recent years because of its pleiotropic biological activities, but considerably fewer studies have been published addressing its role in serum lipids and atherosclerosis compared to other topics covered. AIMS OF THE STUDY: The aim of the present study was to assess the effects of the trans-10,cis-12 isomer of CLA on cholesterolaemia and on several metabolic pathways involved in cholesterol metabolism in hamsters. METHODS: Animals were fed atherogenic diets supplemented with 0.5% linoleic acid, 0.5% trans-10,cis-12 CLA or 1.0% trans-10,cis-12 CLA, for 6 weeks. Serum lipoproteins were separated by FPLC. Cholesterol in serum and liver, as well as triacylglycerols and phospholipids in liver were assessed by spectrophotometry. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR), acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolase (CEH) activities were measured by radiometry, and LDL receptors were determined by Western blot. RESULTS: trans-10,cis-12 CLA feeding did not modify food intake nor final body weight. Although serum total cholesterol remained unchanged, when cholesterol fractions were analyzed a significant decrease in VLDL-cholesterol was observed in CLA-fed animals, without changes in HDL-cholesterol or LDL-cholesterol. trans-10,cis-12 CLA decreased cholesterol ester content and increased free cholesterol in liver. The activity of HMGCoAR was not modified. In contrast, ACAT activity was reduced by both CLA doses and CEH was increased by the high CLA dose. LDL receptors were significantly reduced by trans-10,cis-12 feeding when expressed as arbitrary units per mg of protein, however, the total receptor mass remained unchanged. CONCLUSIONS: These results suggest that, under the present experimental conditions, trans-10,cis-12 CLA feeding reduces cholesterol esterification in liver and decreases the minority serum VLDL-cholesterol fraction, but it does not show a hypocholesterolaemic effect. A dose-response effect was not observed.

Glabrol, an acyl-coenzyme A: cholesterol acyltransferase inhibitor from licorice roots.

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) esterifies free cholesterol in the liver and the intestine. It has relations with production of lipoproteins and accumulation of cholesteryl esters of the atheroma. Therefore, ACAT inhibitors may act as antihypercholesterolemic and antiatherosclerotic agents. One isoprenyl flavonoid was isolated from ethanol extract of licorice roots. On the basis of spectral evidences, the compound was identified as glabrol (1). Compound 1 inhibited rat liver microsomal ACAT activity with an IC(50) value of 24.6 microM and decreased cholesteryl ester formation with an IC(50) value of 26.0 microM in HepG2 cells. In addition, 1 showed a non-competitive type of inhibition against ACAT.

4-hydroxycinnamate lowers plasma and hepatic lipids without changing antioxidant enzyme activities.

BACKGROUND/AIMS: The purpose of this study was to investigate the influence of 4-hydroxycinnamate (4-(OH)-C) supplement on the lipid metabolism and antioxidant system of rats fed a high-cholesterol diet. METHODS: Three groups of rats were given a diet containing 1 g cholesterol/kg for 6 weeks. The control group only received a high cholesterol diet, whereas the other two groups received a diet including lovastatin or 4-(OH)-C (0.1 g/100 g). RESULTS: The plasma total cholesterol concentration was significantly lowered by the 4-(OH)-C supplement, whereas the HDL-cholesterol level was higher in this group. The 4-(OH)-C supplement significantly lowered the hepatic cholesterol and triglycerides levels, respectively. Accumulation of hepatic lipid droplet was the highest in control group; however, it was decreased by supplementation of the 4-(OH)-C and the lovastatin. The hepatic HMG-CoA reductase activities were not significantly different between the groups, whereas the ACAT activity was significantly lowered in the lovastatin group. The 4-(OH)-C significantly lowered the hepatic TBARS content. And it did not alter the neutral sterol and total fecal sterol, however, the fecal acidic sterol was higher in the lovastatin and the 4-(OH)-C groups than in the control group. CONCLUSION: These results indicate that 4-(OH)-C was effective in lowering the plasma cholesterol and hepatic lipids.

Dietary conjugated linoleic acid mixture affects the activity of intestinal acyl coenzyme A: cholesterol acyltransferase in hamsters.

The present study was designed to study the mechanisms by which dietary conjugated linoleic acids (CLA) decrease serum cholesterol. Hamsters were fed a semi-synthetic diet containing 1 g cholesterol/kg diet with or without supplementation with 20 g linoleic acid (LA) and 20 g CLA/kg diet. After 8 weeks, serum fasting total cholesterol (TC) and triacylglycerol (TG) were significantly lower in the LA-supplemented and CLA-supplemented groups compared with those of the control (CTL) hamsters. In contrast to LA, CLA significantly lowered hepatic cholesterol but it increased the level of adipose tissue cholesterol, suggesting that the hypocholesterolaemic mechanism of CLA is different from that of LA. CLA decreased the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT) whereas LA had no effect on this enzyme. Consequently, CLA supplementation increased the faecal excretion of total neutral sterols, but it had no or little effect on the faecal acidic sterols. If the ACAT is associated with cholesterol absorption, the part of mechanisms by which CLA decreases serum cholesterol may involve down-regulation of intestinal ACAT activity.

Fatty acid composition and properties of the liver microsomal membrane of rats fed diets enriched with cholesterol.

Male rats were fed diets containing olive (OO) or evening primrose (EPO) oil (10% w/w), with or without added cholesterol (1% w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in delta 9 and delta 6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased delta 9 desaturase activity as well as membrane microviscosity, whereas it decreased delta 6 and delta 5 desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA:cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups.(ABSTRACT TRUNCATED AT 250 WORDS)