Influence of large molecular polymeric pigments isolated from fermented Zijuan tea on the activity of key enzymes involved in lipid metabolism in rat.

Influence of large molecular polymeric pigments (LMPP) isolated from fermented Zijuan tea on the activity and mRNA expression of key enzymes involved in lipid metabolism in rat was explored. The results show that intragastric infusion of high-dose LMPP (1.215 g/kg body weight) effectively suppressed the elevation in TC and LDL-C (p<0.05), and prevented the reduction in HDL-C (p<0.05), compared with the hyperlipidemia model group. LMPP significantly enhanced the activity of HL and HSL, and increased the HSL mRNA expression in the liver tissue and adipose tissue. High-LMPP treatment significantly reduced the HMG-CoA reductase expression by 56.5% in the liver compared with hyperlipidemia model group. In contrast, LDL-R expression was increased by 120% in the presence of high-LMPP treatment. These results suggest that LMPP have the hypolipidemic effect to some extent and significantly enhance HSL mRNA expression in the liver and adipose tissue, thereby increasing HSL activity in rat.

Indices of reverse cholesterol transport in subjects with metabolic syndrome after treatment with rosuvastatin.

OBJECTIVE: The effects of the statin, rosuvastatin on indices of reverse cholesterol transport were studied in a randomized, placebo-controlled, cross-over trial in 25 overweight subjects with defined metabolic syndrome. RESULT: Four weeks' treatment with 40 mg/day rosuvastatin significantly reduced levels of plasma cholesterol (44%), LDL cholesterol (60%) and triglyceride (38%). HDL cholesterol (mean [S.D.]) rose (0.97[0.17] to 1.05[0.17]mmol/L; P<0.05) and the LpA-I component of HDL from 39[7] to 45[9]mg/dL (P<0.05). LCAT activity fell (0.55[0.13] to 0.35[0.07]nmol/mL/h; P<0.05); CETP activity and mass fell from 89[13] to 80[11]nmol//L/h and from 1.66[0.57] to 1.28[0.41]mug/mL respectively, (P<0.05). Cholesterol efflux in vitro (to plasmas from THP-1 activated cells) fell from 7.1[1.8]% (placebo) to 6.2[1.7]% (rosuvastatin); P<0.05, but when plasmas depleted of apoB lipoproteins were studied, the difference in efflux was no longer statistically significant. During placebo efflux was paradoxically inversely correlated with HDL-C (P=0.016) and LpA-I (P=0.035) concentrations but these correlations were absent after rosuvastatin. CONCLUSIONS: The data suggest possible HDL dysfunctionality in subjects with metabolic syndrome. The reduced capacity of plasmas following statin treatment to stimulate cholesterol efflux in vitro occurred in association with reduction in apoB lipoproteins and reduced activities of CETP and LCAT, and despite increased levels of HDL cholesterol.

Fenofibrate reverses the decline in HDL cholesterol in mice overexpressing human phospholipid transfer protein.

In humans, fibrates are used to treat dyslipidemia, because these drugs lower plasma triglycerides and raise HDL cholesterol. Treatment with fibrates lowers plasma phospholipid transfer protein (PLTP) activity in humans, but increases PLTP activity in mice, without a consistent effect on HDL-cholesterol concentration. Earlier, we found that PLTP overexpression in transgenic mice results in decreased plasma HDL levels and increased diet-induced atherosclerosis. So it seems that the interplay between fibrates, PLTP and HDL is different in mice and man, which may be important for atherosclerosis development. In the present study, we measured the effects of fibrates on PLTP expression in cultured human hepatocytes and effects of fibrate treatment on human PLTP expression, plasma PLTP activity and HDL levels in human PLTP transgenic mice. Fibrate treatment did not influence PLTP mRNA levels in human hepatocytes. Hepatic human PLTP mRNA levels and PLTP activity were both moderately elevated by fenofibrate treatment in human PLTP transgenic mice. In wild-type mice, however, feeding fenofibrate resulted in a strong induction of PLTP mRNA in the liver and a more than 4-fold increase of plasma PLTP activity. Plasma triglycerides were reduced in all mice by 48% or more by fenofibrate treatment. HDL-cholesterol concentrations were substantially increased by fenofibrate in PLTP overexpressing mice (+72%), but unaffected in wild-type mice. We conclude that fenofibrate treatment reverses the HDL-lowering effect of PLTP overexpression in human PLTP transgenic mice.

Lecithin-cholesterol acyltransferase activity during maturation of human preovulatory follicles with different concentrations of ascorbate, alpha-tocopherol and nitrotyrosine.

The enzyme lecithin-cholesterol acyltransferase (LCAT) transfers an acyl chain from lecithin to cholesterol or oestradiol, thus playing a crucial role in reverse cholesterol transport and follicular synthesis of potent long-lived oestrogens. The mechanism of catalysis is biphasic, as it is based on a phospholipase and an esterifying activity. Sulfhydryl groups were previously reported to be required for the esterification step. Lecithin-cholesterol acyltransferase has previously been shown to be inhibited by thiol oxidants such as peroxynitrite. Peroxynitrite also converts tyrosine to nitrotyrosines. In the present study, high levels of nitrotyrosine associated with low LCAT activity, and vice versa, were found in human preovulatory follicular fluids. Follicular fluids were also analysed for oestradiol (E) and progesterone (P) concentrations. The E/P ratio, which decreases as ovulation approaches, was used to evaluate the maturation status of each follicle. Enzyme activity was negatively correlated with the E/P ratio. Ascorbate (Asc) and alpha-tocopherol (Toc) were titrated in follicular fluid and plasma to evaluate their accumulation or consumption in the follicle. High LCAT activity was found in follicular fluids where Asc and Toc had accumulated, whereas lower activity was associated with Asc and Toc consumption. The consumption of both antioxidants was positively correlated with the E/P ratio. The results suggest that as follicle maturation progresses, Toc and Asc concentrations increase in follicular fluid, thus protecting LCAT from oxidative damage and loss of activity.

Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545-552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH(2) (Ac-18A-NH(2) or 2F) were: 3F(3)(Ac-F(3)18A-NH(2)), 3F(14)(Ac-F(14)18A-NH(2)), 4F(Ac-F(3,14)18A-NH(2)), 5F(Ac-F(11,14,17) 18A-NH(2)), 6F(Ac-F(10,11,14,17)18A-NH(2)), and 7F(Ac-F(3,10,11,14,17) 18A-NH(2)). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity. These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.

Lipid-free apolipoproteins A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase.

We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-II on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (LCAT). First, homogeneous rHDL were prepared with either apoA-I or apoA-II using palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. Lipid-free apoA-I and apoA-II were labeled with the fluorescent probe dansyl chloride (DNS). The binding kinetics of apoA-I-DNS to A-II-POPCrHDL and of apoA-II-DNS to A-I-POPCrHDL were monitored by fluorescence polarization, adding the lipid-free apolipoproteins to the rHDL particles in a 1:1 molar ratio. For both apolipoproteins, the binding to rHDL was rapid, occurring within 5 min. Next, the effect on rHDL structure and particle size was determined after incubations of lipid-free apolipoproteins with homogeneous rHDL at 37 degrees C from 0.5 to 24 h. The products were analyzed by non-denaturing gradient gel electrophoresis followed by Western blotting. The effect of apoA-I or apoA-II on 103 A A-II-POPCrHDL was a rearrangement into 78 A particles containing apoA-I and/or apoA-II, and 90 A particles containing only apoA-II. The effect of apoA-I or apoA-II on 98 A A-I-POPCrHDL was a rearrangement into complexes ranging in size from 78 A to 105 A containing apoA-I and/or apoA-II, with main particles of 78 A, 88 A, and 98 A. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addition of apoA-I to A-II-POPCrHDL increased its reactivity with LCAT 24-fold, reflected by a 4-fold increase in apparent V(m)ax and a 6-fold decrease in apparent K(m), while the addition of apoA-II to A-II-POPCrHDL had no effect on its minimal reactivity with LCAT. In contrast, the addition of apoA-II to A-I-POPCrHDL decreased the reaction with LCAT by about one-half. The inhibition was due to a 2-fold increase in apparent K(m); there was no significant change in apparent V(m)ax. Likewise, the addition of apoA-I to A-I-POPCrHDL inhibited the reaction with LCAT to about two-thirds that of A-I-POPCrHDL without added apoA-I. In summary, both lipid-free apoA-I and apoA-II can promote the remodeling of rHDL into hybrid particles of primarily smaller size. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when added to the reaction in lipid-free form. These results have important implications for the roles of lipid-free apoA-I and apoA-II in HDL maturation and metabolism.

Cholesterol-lowering effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on cholesterol diet-fed rats and rabbits.

Pharmacological characterization of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was performed with both in vitro and in vivo assay systems. NTE-122 inhibited microsomal ACAT activities of various tissues (liver of rabbit and rat, small intestine of rabbit and rat, and aorta of rabbit) and cultured cells (HepG2 and CaCo-2), with IC50 values from 1.2 to 9.6 nM. The inhibition mode of NTE-122 was competitive for HepG2 ACAT. NTE-122 had no effect on other lipid metabolizing enzymes, such as 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA synthetase, cholesterol esterase, lecithin:cholesterol acyltransferase, acyl-CoA:sn-glycerol-3-phosphate acyltransferase and cholesterol 7alpha-hydroxylase up to 10 microM. When NTE-122 was administered to the cholesterol diet-fed rats, serum and liver cholesterol levels were markedly reduced with an ED50 of 0.12 and 0.44 mg/kg/day, respectively. In the cholesterol diet-fed rabbits, NTE-122 significantly lowered plasma and liver cholesterol levels at more than 2 mg/kg/day. These results indicate that NTE-122 is a potent, selective and competitive inhibitor of ACAT, making it a worth while therapeutic agent for hypercholesterolemia and atherosclerosis.

Effect of nicotine on lipoprotein metabolism in rats.

Nicotine, a major component of cigarette smoke, plays an important role in the development of cardiovascular disease and lung cancer in smokers. The effect of nicotine on lipoprotein metabolism was studied using rats as the experimental animal. There was a significant increase in the total cholesterol, phospholipids, and triglycerides as well as the amount of lipids associated with very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in sera of nicotine-treated rats. The incorporation of 3H labeled leucine into the apo B was found to be increased both in the medium and associated cells in the hepatocytes isolated from nicotine-treated rats indicating an increased synthesis and secretion of the apo B containing lipoproteins. This was further confirmed by the higher incorporation of 14C acetate into total and individual lipids of LDL and VLDL secreted into the medium as well as that associated with different lipids in the cell layer. The activity of lipoprotein lipase in extrahepatic tissues and plasma lecithin cholesterol acyl transferase activity were significantly lower in nicotine-treated rats. These results indicate that nicotine exerts hyperlipidemic effects particularly by increasing the synthesis and secretion of triglyceride-rich lipoproteins. Since nicotine is one of the major hazardous components present in cigarette smoke and tobacco, one can extrapolate that the deleterious effect exerted by nicotine on rats extends to cigarette smokers and those who use other forms of tobacco.

Effect of selenium on lipids and some lipid metabolising enzymes in DMBA induced mammary tumor rats.

Current evidences clearly point out that an increase in lipid peroxidation influences lipid metabolism in cancer patients. Several investigations recognize selenium as a potent antioxidant, as well as an anticarcinogen, in both animal and human systems. Selenium was administered to Wistar rats bearing mammary tumor induced by 7,12-dimethylbenz(a)anthracene (DMBA) to study alterations in the concentration of lipid profiles and in the activities of some lipid metabolising enzymes. Control and tumor-bearing rats administered with selenium, were fed 5 mg sodium selenite/kg diet from the day of tumor induction. Plasma total lipids, total cholesterol, free fatty acids, triglycerides, phospholipids, VLDL and LDL cholesterol were significantly lower in selenium-treated rats bearing tumors, whereas, plasma ester cholesterol and HDL cholesterol were significantly greater due to selenium administration in DMBA induced-tumor rats. Total lipase and lecithin: cholesterol acyltransferase registered greater activities in plasma of selenium administered rats with tumor, while the activity of preheparin lipoprotein lipases in plasma of rats bearing tumors was lower due to selenium administration. These observations clearly indicate the effect of selenium in correcting the abnormalities of lipid metabolism in tumor-induced rats.

Lipoprotein pattern and plasma lecithin cholesterol acyl transferase activity in children with Alagille syndrome.

Alagille syndrome is frequently associated with hyperlipidemia and xanthoma. The aim of the study was to assess the lipid profile (plasma lipoproteins, apolipoproteins (apo)) and lecithin cholesterol acyl transferase (LCAT) activity, with and without treatment with cholestyramine in Alagille syndrome. Five children (mean age = 6 +/- 4 years) with Alagille syndrome were studied at two different times while receiving no treatment, and while receiving cholestyramine. They were compared with 12 normal controls, who were not different from patients for age and sex. In Alagille syndrome, total serum cholesterol, triglycerides and phospholipids were elevated compared with the controls (P < 0.008). VLDL-cholesterol, LDL-cholesterol, HDL-triglycerides, LDL-triglycerides and VLDL-phospholipids were higher, whereas HDL-cholesterol was lower than controls (P < 0.03). Apo B, CIII, E and lipoprotein particles Lp AI were higher (P < 0.001), whereas Lp AI:AII was lower than controls (P < 0.03). Lipoprotein-X was present in the 5 children with Alagille syndrome and explained in part the elevation of plasma cholesterol, phospholipids, and apo CIII. LCAT activity was decreased (P < 0.01) and might cause some abnormalities of HDL with lower cholesterol, higher triglycerides, apo E and apo CIII contents than controls, and abnormalities of VLDL and LDL with higher cholesterol, triglycerides, phospholipids and apo B contents than controls. Some of the risk factors of atherosclerosis were found in Alagille syndrome, namely high levels of plasma cholesterol, LDL cholesterol, apo B, apo B/apo AI. Treatment with cholestyramine resulted in a few modifications to the lipid profile, while lipoprotein-X and the decrease of LCAT activity persisted.

Enhancement of aortic cholesterol deposition by dietary linoleic acid in cholesterol-fed mice: an animal model for primary screening of antiatherosclerotic agents.

We tried to develop an experimental model using mice for the primary screening of antiatherosclerotic agents. Male ICR strain mice were given a high-cholesterol diet supplemented with 10% linoleic acid for 14 weeks. Throughout the experimental period, weight gain of these mice was significantly inhibited as compared to that of control mice given a basal diet, but displayed a steady increase comparable to that of the high-cholesterol diet without linoleic acid. The cholesterol and linoleic acid-fed mice showed increased serum cholesterol and phospholipid levels, and decreased serum triglyceride and high-density lipoprotein-(HDL) cholesterol levels and lecithin/cholesterol acyltransferase (LCAT) activity, as well as a markedly increased lipid peroxide level which was a characteristic appearance in the serum of this mouse model. At the end of the experiment, uniform and significant increases in cholesterol, notably cholesteryl ester, were observed in the aorta. Also found were marked decreases in the aorta contents of desmosine and isodesmosine, which are cross-linking amino acids present only in the elastin. Histological observations showed accumulations of fatty droplets in the intima. These changes were much less in mice receiving a high-cholesterol diet without linoleic acid. In this mouse model, probucol prevented elevation of serum cholesterol, phospholipid, and cholesterol accumulation in the aorta. Increases in lipid peroxide level and decreases in LCAT activity were also prevented. These findings indicate that this mouse model is useful for primary screening of antiatherosclerotic agents with antioxidative activity.

Effects of probucol on plasma lipids, lipoproteins and parameters of high density lipoprotein metabolism.

Eight patients with primary hypercholesterolemia were treated with probucol for 17 weeks. Plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, and high density lipoprotein (HDL)-cholesterol decreased by 16.6, 15.0 and 25.7%, respectively, in response to probucol treatment. Plasma levels of apolipoprotein B and apolipoprotein A-I also decreased, while apolipoprotein A-II concentrations were unchanged. The decrease in HDL-cholesterol levels was associated with a reduction in HDL particle size. No changes in the plasma lecithin:cholesterol acyltransferase activity or mass occurred in response to probucol treatment. In contrast, a significant 25% increase in plasma cholesteryl ester and triglyceride transfer activity occurred following probucol treatment. There was a positive correlation (R = 0.94) between cholesterol ester and triglyceride transfer. We propose that the increase in lipid transfer activity may in part explain the changes in HDL concentration and size, as well as the previously reported effect probucol has on reducing atherosclerosis in animal models.