Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

l-Alanine-α-Ketobutyrate Aminotransferase of Pseudomonas sp.

Modified fungal product 4-O-methylascochlorin (MAC) is an experimental agent affecting lipid and carbohydrate metabolism in mammals. The hypocholesterolemic properties of MAC were studied using rats fed on a standard laboratory diet. Because of the insolubility in water, reproducibility of the hypocholesterolemic activity had usually been poor for rats fed ad libitum. The difficulty was overcome by controlled reverse-phase feeding; MAC significantly lowered serum total cholesterol (s-TC) in rats only when given by gastric intubation soon after diet intake.

MAC increased fecal excretion of neutral and acidic sterols and also increased biliary flow accompanying increments in biliary cholesterol, bile acids and phospholipids. A much larger increase in neutral sterols was characteristic for MAC. However, intestinal absorption of cholesterol and cholic acid was unaffected by MAC. Three mechanisms therefore seemed to be working in hypocholesterolemic activity: (a) withdrawal of hepatic cholesterol into bile, (b) a larger fecal loss of sterols following increment of biliary sterols and (c) enhanced bile acid synthesis compensating the larger fecal loss. A negative sterol balance often leads to an increase in hepatic cholesterogenesis. However, cholesterogenesis, as judged from incorporation of the precursors, was unchanged by MAC.     

Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol.

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.     

Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice

The scavenger receptor class B type I (SR-BI), which is expressed in the liver and intestine, plays a critical role in cholesterol metabolism in rodents. While hepatic SR-BI expression controls high density lipoprotein (HDL) cholesterol metabolism, intestinal SR-BI has been proposed to facilitate cholesterol absorption. To evaluate further the relevance of SR-BI in the enterohepatic circulation of cholesterol and bile salts, we studied biliary lipid secretion, hepatic sterol content and synthesis, bile acid metabolism, fecal neutral sterol excretion, and intestinal cholesterol absorption in SR-BI knockout mice. SR-BI deficiency selectively impaired biliary cholesterol secretion, without concomitant changes in either biliary bile acid or phospholipid secretion. Hepatic total and unesterified cholesterol contents were slightly increased in SR-BI-deficient mice, while sterol synthesis was not significantly changed. Bile acid pool size and composition, as well as fecal bile acid excretion, were not altered in SR-BI knockout mice. Intestinal cholesterol absorption was somewhat increased and fecal sterol excretion was slightly decreased in SR-BI knockout mice relative to controls. These findings establish the critical role of hepatic SR-BI expression in selectively controlling the utilization of HDL cholesterol for biliary secretion. In contrast, SR-BI expression is not essential for intestinal cholesterol absorption.     

Effect of diosgenin on lipid metabolism in rats.

The purpose of this study was to determine whether diosgenin suppresses cholesterol absorption in rats, and to examine relevant changes in cholesterol and bile acid metabolism. Diosgenin fed with the diet for 1 week inhibited cholesterol absorption as determined by the serum isotope ratio technique, as well as by measuring in the feces the amount of unabsorbed radioactivity from orally administered [3H]cholesterol. In addition, diosgenin suppressed the serum and liver uptake of radioactivity from co-administered [3H]cholesterol as well as the accumulation of liver cholesterol in the cholesterol-fed rat; diosgenin was substantially more active than cholestyramine or beta-sitosterol. In vitro, diosgenin had no effect on the activity of rat pancreatic esterase. Diosgenin decreased the elevated cholesterol in serum LDL and elevated cholesterol in the HDL fraction of cholesterol-fed rats; diosgenin had no effect on serum cholesterol in normocholesterolemic rats. In contrast to cholestyramine, diosgenin markedly increased neutral sterol excretion without altering bile acid excretion; in vitro, diosgenin had no effect on bile acid binding. Diosgenin treatment increased hepatic and intestinal cholesterol synthesis as well as the activity of hepatic HMG CoA reductase. This was accompanied by increased biliary concentration of cholesterol, but not of bile acids. Diosgenin had no effect on cholesterol synthesis when added to normal rat liver homogenates. It was concluded that diosgenin interferes with the absorption of cholesterol of both exogenous and endogenous origin; such interference is accompanied by derepressed, i.e., increased, rates of hepatic and intestinal cholesterol synthesis. The increased unabsorbed cholesterol together with enhanced secretion of cholesterol into bile resulted in increased excretion of neutral sterols without affecting the biliary and fecal excretion of bile acids.     

Biliary lipid secretion in hypercholesterolemia.

A report on the effects of primary bile acid ingestion alone or in combination with plant sterols on serum cholesterol levels, biliary lipid secretion, and bile acid metabolism. Biliary bile acid and cholesterol secretion were measured in four patients with type IIa hypercholesterolemia before and after randomized treatment periods. During these periods either a bile acid mixture (cholic-chenodeoxycholic 2:1, a proportion similar to that endogenously synthesized in health), at a level of 20 mg/kg, or the same mixture plus sitosterols, 200 mg/kg, was fed. Serum cholesterol and the cholesterol saturation of fasting-state bile was also measured. Pretreatment biliary lipid secretion was within normal limits. Bile acid kinetic measurements were also recorded before treatment and showed that cholic acid synthesis was disproportionately decreased relative to that of chenodeoxycholic acid, a finding previously reported by others. Administration of the bile acid mixture increased biliary bile acid secretion in 3 of 4 patients, but did not influence biliary cholesterol secretion. The combination of sitosterol-bile acid, however, caused a relative decrease in cholesterol secretion in bile, and fasting-state bile became unsaturated in all patients. No change in fecal neutral sterol excretion occurred during the beta-sitosterol-bile acid regimen, suggesting that simultaneous bile acid feeding blocks the compensatory increase in cholesterol synthesis known to be induced by beta-sitosterol feeding in hypercholesterolemic patients. Serum cholesterol levels also fell modestly during the sitosterol-bile acid regimen, the decrease averaging 15%. We conclude that the abnormally low rate of bile acid synthesis in patients with type IIa hyperlipoproteinemia does not influence biliary lipid secretion; that increasing the input of the two primary bile acids into the enterohepatic circulation does not increase biliary cholesterol secretion or lower serum cholesterol levels in such patients; and that the usual increase in cholesterol synthesis induced by beta-sitosterol feeding does not occur if bile acids are administered simultaneously.     

Antilithiasic effect of beta-cyclodextrin in LPN hamster: comparison with cholestyramine

Beta-Cyclodextrin (BCD), a cyclic oligosaccharide that binds cholesterol and bile acids in vitro, has been previously shown to be an effective plasma cholesterol lowering agent in hamsters and domestic pigs. This study examined the effects of BCD as compared with cholestyramine on cholesterol and bile acid metabolism in the LPN hamster model model for cholesterol gallstones. The incidence of cholesterol gallstones was 65% in LPN hamsters fed the lithogenic diet, but decreased linearly with increasing amounts of BCD in the diet to be nil at a dose of 10% BCD. In gallbladder bile, cholesterol, phospholipid and chenodeoxycholate concentrations, hydrophobic and lithogenic indices were all significantly decreased by 10% BCD. Increases in bile acid synthesis (+110%), sterol 27-hydroxylase activity (+106%), and biliary cholate secretion (+140%) were also observed, whereas the biliary secretion of chenodeoxycholate decreased (-43%). The fecal output of chenodeoxycholate and cholate (plus derivatives) was increased by +147 and +64%, respectively, suggesting that BCD reduced the chenodeoxycholate intestinal absorption preferentially. Dietary cholestyramine decreased biliary bile acid concentration and secretion, but dramatically increased the fecal excretion of chenodeoxycholate and cholate plus their derivatives (+328 and +1940%, respectively). In contrast to BCD, the resin increased the lithogenic index in bile, induced black gallstones in 34% of hamsters, and stimulated markedly the activities of HMG-CoA reductase (+670%), sterol 27-hydroxylase (+310%), and cholesterol 7alpha-hydroxylase (+390%). Thus, beta-cyclodextrin (BCD) prevented cholesterol gallstone formation by decreasing specifically the reabsorption of chenodeoxycholate, stimulating its biosynthesis and favoring its fecal elimination. BCD had a milder effect on lipid metabolism than cholestyramine and does not predispose animals to black gallstones as cholestyramine does in this animal model.     

Effect of dietary inclusion of Lactobacillus acidophilus ATCC 43121 on cholesterol metabolism in rats

This study examined the effects of Lactobacillus acidophilus ATCC 43121 (LAB) on cholesterol metabolism in hypercholesterolemia-induced rats. Four treatment groups of rats (n = 9) were fed experimental diets: normal diet, normal diet+LAB (2 x 10(6) CFU/day), hypercholesterol diet (0.5% cholesterol, w/w), and hypercholesterol diet + LAB. Body weight, feed intake, and feed efficiency did not differ among the four groups. Supplementation with LAB reduced total serum cholesterol (25%) and VLDL + IDL + LDL cholesterol (42%) in hypercholesterol diet groups, although hepatic tissue cholesterol and lipid contents were not changed. In the normal diet group, cholesterol synthesis (HMG-CoA reductase expression), absorption (LDL receptor expression), and excretion via bile acids (cholesterol 7 alpha-hydroxylase expression) were increased by supplementation with LAB, and increased cholesterol absorption and decreased excretion were found in the hypercholesterol diet group. Total fecal acid sterols excretion was increased by supplementation with LAB. With proportional changes in both normal and hypercholesterol diet groups, primary bile acids (cholic and chenodeoxycholic acids) were reduced, and secondary bile acids (deoxycholic and lithocholic acids) were increased. Fecal neutral sterol excretion was not changed by LAB. In this experiment, the increase in insoluble bile acid (lithocholic acid) reduced blood cholesterol level in rats fed hypercholesterol diets supplemented with LAB. Thus, in the rat, L. acidophilus ATCC 43121 is more likely to affect deconjugation and dehydroxylation during cholesterol metabolism than the assimilation of cholesterol into cell membranes.     

Sialic acid content of low density lipoprotein and its relation to lipid concentrations and metabolism of low density lipoprotein and cholesterol

A low sialic acid content in low density lipoprotein (LDL) has been associated with atherogenicity and coronary artery disease (CAD) in many but not all studies. We investigated associations of the sialic acid-to-apolipoprotein B (apoB) ratio of LDL with lipoprotein lipid concentrations, kinetics of LDL, metabolism of cholesterol, and the presence of CAD in 98 subjects (CAD(+), n = 56; CAD(-), n = 42). The sialic acid ratios of total, dense, and very dense LDL were lower in the CAD(+) than CAD(-) subjects, especially at high sialic acid ratios. The LDL sialic acid ratio was inversely associated with respective lipid and apoB concentrations and positively with lipid-to-apoB ratios of LDL. The transport rates (TRs) for total and dense LDL apoB were negatively associated with their sialic acid ratios. The sialic acid ratio of dense LDL, but not that of total LDL, was inversely correlated with serum levels of cholesterol precursor sterols, indicators of cholesterol synthesis, and positively with serum levels of plant sterols, indicators of cholesterol absorption. In addition, the TR for dense LDL was positively correlated with cholesterol synthesis.In conclusion, a low LDL sialic acid ratio was associated with CAD, high numbers of small LDL particles, and a high TR for LDL apoB, and in dense LDL also with high synthesis and low absorption of cholesterol.     

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.     

Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells

The effects of ketoconazole, a lanosterol demethylase and cytochrome P450 inhibitor, on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity and sterol biosynthesis were studied in rat intestinal epithelial cell cultures (IEC-6). Incubation of cells with 0.15-2 microM ketoconazole resulted in a concentration-dependent inhibition of reductase activity. As the drug concentration approached 15 microM, the reductase activity returned to control values, and at 30 microM ketoconazole, a stimulation of enzyme activity was observed. The drug had no effect on reductase activity in homogenates of IEC-6 cells. Ketoconazole (0.15-30 microM) caused a concentration-dependent inhibition of the incorporation of [3H] mevalonolactone into cholesterol with a concomitant accumulation of radioactivity in methyl sterols; e.g. lanosterol and 24,25-epoxylanosterol. Interestingly, the incorporation of radioactivity into polar sterols showed a biphasic response which was inversely proportional to the biphasic response of reductase activity. Thus, incorporation of [3H]mevalonolactone into polar sterols increased at low concentrations of ketoconazole (0.15-2 microM) and decreased to control values at high concentrations of the drug. Treatment of cells with ketoconazole (30 microM) and [3H]mevalonolactone followed by removal of the drug and radiolabel resulted in an inhibition of reductase activity and a redistribution of radioactivity from lanosterol and 24,25-epoxylanosterol to cholesterol and polar sterols. These results suggested that the inhibition of reductase activity at low concentrations of ketoconazole (less than 2 microM) was due to a formation of regulatory polar sterols generated from the methyl sterols. At high concentrations of ketoconazole (30 microM) where no suppression in reductase activity was observed, the conversion of exogenously added [3H]24(S),25-epoxylanosterol to polar sterols was prevented. Exogenously added 24,25-epoxylanosterol inhibited reductase activity in a dose-dependent fashion, and ketoconazole (30 microM) prevented the inhibition caused by low concentrations of epoxylanosterol. The drug, however, was unable to prevent the dose-dependent suppression of reductase activity by 25-hydroxylanosterol, a reduced form of 24,25-epoxylanosterol. These results indicated that 24,25-epoxylanosterol per se was not an inhibitor of reductase activity but could be metabolized to regulatory polar sterols through a cytochrome P-450 dependent reaction which was sensitive to ketoconazole. Treatment of cells with ketoconazole totally abolished the inhibition of reductase activity by low density lipoprotein (LDL).(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of cholesterol metabolism by dietary plant sterols

Renewal has occurred in the use of plant sterols for the treatment of hypercholesterolemias. A novel development was to convert plant sterols to corresponding stanols and esterify them to fat soluble form. In contrast to the crystalline plant sterols or stanols, plant stanol esters can be easily consumed during normal food intake in soluble form in different fat-containing food constituents when they have a potent cholesterol-lowering effect, shown in normo- and hypercholesterolemic men and women without or with coronary heart disease, children and diabetes. Cholesterol lowering is approximately 10% for total and 15% for LDL cholesterol, with the respective values for stanol ester margarine (2-3 g/day stanols) being 15% and 20%. Stanol esters reduce cholesterol absorption efficiency by up to 65%, increase cholesterol elimination in feces as cholesterol itself, usually not as bile acids, and stimulate cholesterol synthesis. Serum beta-carotene level is lowered, but no fat malabsorption or lowering of serum fat soluble vitamins have been observed. In contrast to plant sterols, stanols and their esters are minimally absorbed and they reduce serum plant sterol concentrations, also preventing statin-induced increase of plant sterols. Stanol ester margarine has been included in dietary treatment of hypercholesterolemia followed by the addition of drug treatment in resistant cases.     

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

Bile acid synthesis precursors in familial combined hyperlipidemia: The oxysterols 24S-hydroxycholesterol and 27-hydroxycholesterol

Familial combined hyperlipidemia (FCHL), the most common inherited disorder of lipid metabolism is characterized by increasing cholesterol synthesis precursors due to hepatic overproduction of cholesterol. The bile acids synthesis pathway has not been previously studied in FCHL. The aim of this work was to study the oxysterol levels which are involved in the bile acids synthesis from cholesterol in FCHL. Clinical parameters and subclinical atherosclerosis were studied in a total of 107 FCHL patients and 126 normolipidemic controls. Non cholesterol sterols (desmosterol and lanosterol) and oxysterols (27-hydroxycholesterol and 24S-hydroxycholesterol) were measured by high performance liquid chromatography tandem mass spectrometry. Desmosterol and lanosterol, markers of cholesterol synthesis, had a positive correlation with BMI and apo B. However, no correlation was found for 24S-hydroxycholesterol and 27-hydroxycholesterol, precursors of bile acids, with these clinical parameters. Only 27-hydroxycholesterol had a positive correlation with apo B, ρ = 0.204 (P = 0.037). All oxysterol levels were higher in FHCL as compared to normal controls. A total of 59 FCHL subjects (59%) presented values of 24S-hydroxycholesterol above the 95th percentile of this oxysterol in the control population. All oxysterols showed no association with fat mass in contrast with non-cholesterol sterols. FCHL subjects with oxysterol overproduction had less carotid intima media thickness (cIMT), which suggests less atherosclerosis in these subjects. In summary, our data indicate that high oxysterol levels might be good markers of FCHL, unrelated to fat mass, and may exert a protective mechanism for cholesterol accumulation.     

Hepatic SR-BI, not endothelial lipase, expression determines biliary cholesterol secretion in mice

High density lipoprotein cholesterol is thought to represent a preferred source of sterols secreted into bile following hepatic uptake by scavenger receptor class B type I (SR-BI). The present study aimed to determine the metabolic effects of an endothelial lipase (EL)–mediated stimulation of HDL cholesterol uptake on liver lipid metabolism and biliary cholesterol secretion in wild-type, SR-BI knockout, and SR-BI overexpressing mice. In each model, injection of an EL expressing adenovirus decreased plasma HDL cholesterol (P < 0.001) whereas hepatic cholesterol content increased (P < 0.05), translating into decreased expression of sterol-regulatory element binding protein 2 (SREBP2) and its target genes HMG-CoA reductase and LDL receptor (each P < 0.01). Biliary cholesterol secretion was dependent on hepatic SR-BI expression, being decreased in SR-BI knockouts (P < 0.001) and increased following hepatic SR-BI overexpression (P < 0.001). However, in each model, biliary secretion of cholesterol, bile acids, and phospholipids as well as fecal bile acid and neutral sterol content, remained unchanged in response to EL overexpression. Importantly, hepatic ABCG5/G8 expression did not correlate with biliary cholesterol secretion rates under these conditions. These results demonstrate that an acute decrease of plasma HDL cholesterol levels by overexpressing EL increases hepatic cholesterol content but leaves biliary sterol secretion unaltered. Instead, biliary cholesterol secretion rates are related to the hepatic expression level of SR-BI. These data stress the importance of SR-BI for biliary cholesterol secretion and might have relevance for concepts of reverse cholesterol transport.     

Inhibiting intestinal NPC1L1 activity prevents diet-induced increase in biliary cholesterol in Golden Syrian hamsters

Niemann-Pick C1-like 1 (NPC1L1) facilitates the uptake of sterols into the enterocyte and is the target of the novel cholesterol absorption inhibitor, ezetimibe. These studies used the Golden Syrian hamster as a model to delineate the changes in the relative mRNA expression of NPC1L1 and other proteins that regulate sterol homeostasis in the enterocyte during and following cessation of ezetimibe treatment and also to address the clinically important question of whether the marked inhibition of cholesterol absorption alters biliary lipid composition. In hamsters fed a low-cholesterol, low-fat basal diet, the abundance of mRNA for NPC1L1 in the small intestine far exceeded that in other regions of the gastrointestinal tract, liver, and gallbladder. In the first study, female hamsters were fed the basal diet containing ezetimibe at doses up to 2.0 mg.day(-1).kg body wt(-1). At this dose, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased by 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition. The ezetimibe-induced changes in intestinal cholesterol handling were reversed when treatment was withdrawn. In a second study, male hamsters were given a diet enriched in cholesterol and safflower oil without or with ezetimibe. The lipid-rich diet raised the absolute and relative cholesterol levels in bile more than fourfold. This increase was largely prevented by ezetimibe. These data are consistent with the recent finding that ezetimibe treatment significantly reduced biliary cholesterol saturation in patients with gallstones.     

A relationship between the activities of hepatic lanosterol 14 alpha-demethylase and 3-hydroxy-3-methylglutaryl-CoA reductase.

At 1-2 h after intragastric administration of ketoconazole, a cytochrome P-450 inhibitor, to rats, there was a 50-60% decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Inhibition reached a maximum at 6-12 h after the drug was given, but after 24 h enzyme activity was stimulated by 60%. The rates of synthesis of hepatic non-saponifiable lipids in vivo showed a similar time-dependent pattern of change. During the first few hours after drug administration, the hepatic cytochrome P-450-dependent metabolism of lanosterol was suppressed in vivo. However, 24 h after treatment, this activity was stimulated, an effect which was also observed by pre-treatment of the rats with the drug for several days. Suppression of hepatic HMG-CoA reductase and lanosterol 14 alpha-demethylase activities was accompanied by a relative increase in the accumulation of labelled polar sterols in the liver in vivo. In the intestine, ketoconazole also resulted in a rapid decline in the rate of synthesis of non-saponifiable lipids and an inhibition of lanosterol 14 alpha-demethylation in vivo. However, in contrast with the liver, there was no stimulation of non-saponifiable lipid synthesis after 24 h.     

Selective sterol accumulation in ABCG5/ABCG8-deficient mice

The ATP binding cassette (ABC) transporters ABCG5 and ABCG8 limit intestinal absorption and promote biliary secretion of neutral sterols. Mutations in either gene cause sitosterolemia, a rare recessive disease in which plasma and tissue levels of several neutral sterols are increased to varying degrees. To determine why patients with sitosterolemia preferentially accumulate noncholesterol sterols, levels of cholesterol and the major plant sterols were compared in plasma, liver, bile, and brain of wild-type and ABCG5/ABCG8-deficient (G5G8(-/-)) mice. The total sterol content of liver and plasma was similar in G5G8(-/-) mice and wild-type animals despite an approximately 30-fold increase in noncholesterol sterol levels in the knockout animals. The relative enrichment of each sterol in the plasma and liver of G5G8(-/-) mice (stigmasterol > sitosterol = cholestanol > bassicasterol > campesterol > cholesterol) reflected its relative enrichment in the bile of wild-type mice. These results indicate that 24-alkylated, Delta22, and 5alpha-reduced sterols are preferentially secreted into bile and that preferential biliary secretion of noncholesterol sterols by ABCG5 and ABCG8 prevents the accumulation of these sterols in normal animals. The mRNA levels for 13 enzymes in the cholesterol biosynthetic pathway were reduced in the livers of the G5G8(-/-) mice, despite a 50% reduction in hepatic cholesterol level. Thus, the accumulation of sterols other than cholesterol is sensed by the cholesterol regulatory machinery.