Short- and long-term effects of biliary drainage on hepatic cholesterol metabolism in the rat.

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.     

Contraceptive steroids increase cholesterol in bile: mechanisms of action

Contraceptive steroids increase the risk of acquiring cholesterol gallstones. The factors responsible include an increase in cholesterol saturation of bile and an increase in rate of secretion of cholesterol into bile. The goal of this study was to investigate the mechanism(s) of these increases in biliary cholesterol. During the use of contraceptive steroids, cholesterol saturation of gallbladder bile and the amount of cholesterol secreted per mole of bile acid increased (P less than 0.05 and P less than 0.02, respectively). Cholesterol absorption, cholesterol synthesis, chylomicron remnant clearance, and the concentration of plasma and lipoprotein lipids were not altered by contraceptive steroids. Despite this apparent lack of effect, important correlations were present during steroid use. LDL (low density lipoprotein) cholesterol increased as dietary cholesterol increased (r = 0.58, P less than 0.025). Cholesterol synthesis correlated directly with VLDL cholesterol concentration (r = 0.64, P less than 0.01), biliary cholesterol secretion (r = 0.68, P less than 0.01) and with molar percent cholesterol in bile (r = 0.49, P = 0.06). Chylomicron remnant clearance also correlated with cholesterol secretion (r = 0.85, P less than 0.001). As either remnant uptake or synthesis increased, the effect of the other source of hepatic cholesterol on biliary cholesterol secretion diminished. These relationships were not observed in the same subjects when they were not taking the hormones. The findings suggest that both newly synthesized and dietary cholesterol contribute to the cholesterol secreted in bile. This is consistent with the hypothesis that cholesterol for secretion into bile and VLDL is derived from a common metabolic pool of free cholesterol. It is proposed that contraceptive steroids exert their effect on biliary cholesterol by increasing cholesterol entering the pool and/or by inhibiting hepatic ACAT (acylcoenzyme A:cholesterol acyltransferase) activity, a known effect of progesterone, so that an increase in free cholesterol entering the pool leads to an increase in output.     

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.     

Flow cytometric assessment of LDL receptor activity in peripheral blood mononuclear cells compared to gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia.

BACKGROUND: Studies indicate that human peripheral blood mononuclear cells mirror low-density lipoprotein (LDL) receptor activity of other cells in the body. To measure LDL receptor activity in patients with heterozygous familial hypercholesterolemia (FH), we prepared peripheral blood mononuclear cells from individuals with molecularly verified LDL receptor defective (Trp66-Gly mutation, n = 18) or receptor negative (Trp23-stop mutation, n = 17) heterozygous FH and from healthy individuals (n = 24). METHODS: The cells were stimulated to express maximum LDL receptor by preincubation in lipoprotein-free medium. They were then incubated at 4 degrees or 37 degrees C with fluorescently conjugated LDL (DiI-LDL). T-lymphocytes and monocytes were identified by fluorescently conjugated monoclonal antibodies. DiI-LDL bound (at 4 degrees C) or internalized (at 37 degrees C) by the cells was measured using flow cytometry. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of our functional assay with the DNA diagnosis. RESULTS: The diagnostic accuracy did not allow our assay to be used for diagnosis of individual cases of heterozygous FH. CONCLUSIONS: We suggest that our two-color fluorescence flow cytometry assay can be used to characterize functionally gene mutations causing LDL receptor dysfunction in patients with heterozygous FH.     

Hepatic lipid accumulation in apolipoprotein C-I-deficient mice is potentiated by cholesteryl ester transfer protein

The impact of apolipoprotein C-I (apoC-I) deficiency on hepatic lipid metabolism was addressed in mice in the presence or the absence of cholesteryl ester transfer protein (CETP). In addition to the expected moderate reduction in plasma cholesterol levels, apoCIKO mice showed significant increases in the hepatic content of cholesteryl esters (+58%) and triglycerides (+118%) and in biliary cholesterol concentration (+35%) as compared with wild-type mice. In the presence of CETP, hepatic alterations resulting from apoC-I deficiency were enforced, with up to 58% and 302% increases in hepatic levels of cholesteryl esters and triglycerides in CETPTg/apoCIKO mice versus CETPTg mice, respectively. Biliary levels of cholesterol, phospholipids, and bile acids were increased by 88, 77, and 20%, respectively, whereas total cholesterol, HDL cholesterol, and triglyceride concentrations in plasma were further reduced in CETPTg/apoCIKO mice versus CETPTg mice. Finally, apoC-I deficiency was not associated with altered VLDL production rate. In line with the previously recognized inhibition of lipoprotein clearance by apoC-I, apoC-I deficiency led to decreased plasma lipid concentration, hepatic lipid accumulation, and increased biliary excretion of cholesterol. The effect was even greater when the alternate reverse cholesterol transport pathway via VLDL/LDL was boosted in the presence of CETP.     

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.     

Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans

Our aim was to identify and quantify the major in vivo pathways of lipoprotein cholesteryl ester transport in humans. Normal (n = 7), bile fistula (n = 5), and familial hypercholesterolemia (FH; n = 1) subjects were studied. Each received isotopic free cholesterol in HDL, LDL, or particulate form, along with another isotope of free or esterified cholesterol or mevalonic acid. VLDL, intermediate density lipoprotein (IDL), LDL, HDL, blood cells, and bile were collected for up to 6 days for analysis of radioactivity and mass of free and esterified cholesterol. These raw data were subjected to compartmental analysis using the SAAM program. Results in all groups corroborated net transport of free cholesterol to the liver from HDL, shown previously in fistula subjects. New findings revealed that 70% of ester was produced from free cholesterol in HDL and 30% from free cholesterol in LDL, IDL, and VLDL. No evidence was found for tissue-produced ester in plasma. There was net transfer of cholesteryl ester to VLDL and IDL from HDL and considerable exchange between LDL and HDL. Irreversible ester output was from VLDL, IDL, and LDL, but very little was from HDL, suggesting that selective and holoparticle uptakes of HDL ester are minor pathways in humans. It follows that 1) they contribute little to reverse transport, 2) very high HDL would not result from defects thereof, and 3) the clinical benefit of high HDL is likely explained by other mechanisms. Reverse transport in the subjects with bile fistula and FH was facilitated by ester output to the liver from VLDL plus IDL.     

Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Processing of cholesteryl ester from low-density lipoproteins in the rat. Hepatic metabolism and biliary secretion after uptake by different hepatic cell types.

Biliary secretion of the cholesteryl ester moiety of (modified) low-density lipoprotein (LDL) was examined under various experimental conditions in the rat. Human LDL or acetylated LDL (acetyl-LDL), radiolabelled with [3H]cholesteryl oleate, was administered intravenously to unanesthetized rats equipped with permanent catheters in the bile duct, duodenum and heart. LDL was cleared relatively slowly from plasma, mainly by Kupffer cells. At 3 h after injection, only 0.9% of the radioactivity was found in bile; after 12 h this value was 4.5%. Uptake of LDL by hepatocytes was stimulated by treatment of the rats with 17 alpha-ethinyloestradiol (EE; 5 mg/kg for 3 successive days); this resulted in a more rapid secretion of radioactivity into bile, 3.9% and 12.4% after 3 h and 12 h respectively. The extremely rapid uptake of acetyl-LDL via the scavenger pathway, mainly by endothelial cells, resulted in the secretion of only 2.1% of its 3H label into bile within 3 h, and 9.5% within 12 h. Radioactivity in bile was predominantly in the form of bile acids; only a small part was secreted as free cholesterol. However, the specific radioactivity of biliary cholesterol was higher than that of bile acids in all three experimental conditions. EE-treated animals did not form cholic acid from [3H]cholesteryl oleate, which was a major product of the cholesteryl oleate from LDL and acetyl-LDL in untreated rats, but formed predominantly very polar bile acids, i.e. muricholic acids. It is concluded that uptake of human LDL or acetyl-LDL by the liver of untreated rats is not efficiently coupled to biliary secretion of cholesterol (bile acids). This might be due to the anatomical localization of their principal uptake sites, the Kupffer cells and the endothelial cells respectively. Induction of LDL uptake by hepatocytes by EE treatment warrants a more efficient disposition of cholesterol from the body via bile.     

Expression of ABCG5 and ABCG8 is required for regulation of biliary cholesterol secretion

The major pathway for elimination of cholesterol in mammals is via secretion into bile. Biliary cholesterol secretion is mediated by the ATP-binding cassette (ABC) transporters ABCG5 (G5) and ABCG8 (G8) and is stimulated by cholesterol and by the non-cholesterol steroids cholate and diosgenin. To define the relationship between G5G8 expression and biliary cholesterol secretion, we measured G5 and G8 mRNA levels and biliary cholesterol concentrations in genetically manipulated mice expressing 0, 1, 2, 5, 10, or 16 copies of the two genes. Biliary cholesterol levels varied directly with G5G8 copy number and hepatic mRNA levels over a >16-fold range. Thus neither delivery of cholesterol to the transporter nor levels of cholesterol acceptors in bile were limiting under these conditions. In wild-type mice, cholate and diosgenin both increased biliary cholesterol concentrations 2-3-fold. The increase in biliary cholesterol content was dependent on expression of G5 and G8; neither steroid increased biliary cholesterol levels in G5G8-/- mice. Cholate treatment was associated with a farnesoid X receptor (FXR)-dependent increase in hepatic mRNA and protein levels of G5 and G8. In contrast to cholate, diosgenin treatment did not affect G5G8 expression. Diosgenin increased the expression of several pregnane X receptor (PXR) target genes and the choleretic effect of diosgenin was reduced by approximately 70% in PXR knock-out mice. Thus G5 and G8 are required to modulate biliary cholesterol secretion in response to cholate and diosgenin, but the choleretic effects of these two steroids are mediated by different mechanisms requiring FXR and PXR, respectively.     

Inheritance pattern of familial hypercholesterolemia and markers of cardiovascular risk

Studies in children and adults have resulted in conflicting evidence in the quest for the answer to the hypothesis that offspring from hypercholesterolemic mothers might have an increased cardiovascular risk. Previous studies might have suffered from limitations such as cohort size and clinical sampling bias. We therefore explored this hypothesis in large cohorts of both subjects with familial hypercholesterolemia (FH) and unaffected siblings in a wide age range. In three cohorts (cohort 1: n = 1,988, aged 0–18 years; cohort 2: n = 300, 8–30 years; cohort 3: n = 369, 18–60 years), we measured lipid and lipoproteins as well as carotid intima-media thickness (c-IMT) in offspring from FH mothers versus FH fathers. For LDL cholesterol, triglycerides (TGs), and c-IMT, we performed a pooled analysis. No significant differences could be observed in c-IMT, lipid, or lipoprotein levels from offspring of FH mothers versus FH fathers. Pooled analyses showed no significant differences for either LDL cholesterol [mean difference 0.02 (−0.06,0.11) mmol/l, P = 0.60], TGs [mean difference 0.07 (0.00,0.14) mmol/l, P = 0.08], or c-IMT [mean difference −0.00 (−0.01,0.01) mm, P = 0.86]. Our data do not support the hypothesis that cardiovascular risk markers are different between offspring from FH mothers and FH fathers.     

Influence of probucol on cholesterol and lipoprotein metabolism in man

The mechanisms for the hypocholesterolemic action of probucol were examined in 17 patients with various levels of plasma cholesterol and triglycerides (TG). All the patients were studied on a metabolic ward. The first period of 6 weeks was for control. Thereafter, probucol was started, and after 2-6 months of drug treatment, the patients were readmitted for another 6-week period for a repeat study. During treatment with probucol, the cholesterol decreased in total plasma by an average of 12%, in low density lipoproteins (LDL) by 11%, and in high density lipoproteins (HDL) by 9%. The TG in total plasma and in very low density lipoproteins (VLDL) remained unchanged during probucol treatment. Turnover of low density lipoprotein apoprotein (apoLDL) was estimated following injection of 125I-labeled apoLDL. Probucol increased the fractional catabolic rate (FCR) for apoLDL by an average of 23%, but did not change apoLDL synthesis. The drug produced no consistent changes in fecal excretion of cholesterol (neutral steroids) and bile acids, in cholesterol absorption, in lipid composition of gallbladder bile, in biliary secretion of cholesterol and bile acids, or in the activities of lipoprotein lipase and hepatic lipase. These data show that probucol lowers LDL by increasing its catabolism. This effect appears to be independent of any changes in metabolism of cholesterol or bile acids.     

Effects of pravastatin and cholestyramine on products of the mevalonate pathway in familial hypercholesterolemia

Patients with heterozygous familial hypercholesterolemia (n = 12) were treated either with pravastatin, a specific inhibitor of HMG-CoA reductase, or cholestyramine, followed by a period of combined treatment with both drugs. Initially, these patients had increased serum levels of low density lipoprotein (LDL) cholesterol (8.77 +/- 0.48 mmol/l; SEM), lathosterol (5.32 +/- 0.60 mg/l), and ubiquinone (0.76 +/- 0.09 mg/l), while the serum dolichol concentration was in the normal range. Cholestyramine treatment (n = 6) decreased the levels of LDL cholesterol (-32%) and increased lathosterol (+125%), but did not change dolichol or ubiquinone levels in a significant manner. Pravastatin treatment (n = 6) decreased LDL cholesterol (-27%), lathosterol (-46%), and ubiquinone (-29%). In this case, the amount of dolichol in serum also showed a small but statistically insignificant decrease (-16%) after 12 weeks of treatment. Combined treatment with cholestyramine and pravastatin (n = 6) resulted in changes that were similar to, but less pronounced than, those observed during pravastatin treatment alone. In no case was the ratio between ubiquinone and LDL cholesterol reduced. Possible effects on hepatic cholesterol, ubiquinone, and dolichol concentrations were studied in untreated (n = 2), cholestyramine-treated (n = 2), and pravastatin-treated (n = 4) gallstone patients and no consistent changes could be observed. The results indicate that treatment with pravastatin in familial hypercholesterolemia decreases serum ubiquinone levels in proportion to the reduction in LDL cholesterol.     

Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile. Dietary garlic and onion have been recently observed to possess anti-lithogenic potential in experimental mice. In this investigation, the role of biliary proteins from rats fed lithogenic diet or garlic/onion-containing diet in the formation of cholesterol gallstones in model bile was studied. Cholesterol nucleation time of the bile from lithogenic diet group was prolonged when mixed with bile from garlic or onion groups. High molecular weight proteins of bile from garlic and onion groups delayed cholesterol crystal growth in model bile. Low molecular weight (LMW) proteins from the bile of lithogenic diet group promoted cholesterol crystal growth in model bile, while LMW protein fraction isolated from the bile of garlic and onion groups delayed the same. Biliary LMW protein fraction was subjected to affinity chromatography using Con-A and the lectin-bound and unbound fractions were studied for their influence on cholesterol nucleation time in model bile. Major portion of biliary LMW proteins in lithogenic diet group was bound to Con-A, and this protein fraction promoted cholesterol nucleation time and increased cholesterol crystal growth rate, whereas Con-A unbound fraction delayed the onset of cholesterol crystallization. Biliary protein from garlic/onion group delayed the crystallization and interfered with pronucleating activity of Con-A bound protein fraction. These data suggest that apart from the beneficial modulation of biliary cholesterol saturation index, these Allium spices also influence cholesterol nucleating and antinucleating protein factors that contribute to their anti-lithogenic potential.     

Reconstitution of LDL with Lipophilic Fluorescein Derivatives: Quantitative Analysis of the Receptor Activity of Human Lymphocytes

Abstract

Low density lipoprotein (LDL), the major cholesterol transport protein in human plasma, consists of an apolar core of cholesteryl esters surrounded by a polar shell containing phospho-lipids, unesterified cholesterol and protein. In the current paper we report the absorption and fluorescence spectra of members of a new class of lipophilic fluorescein derivatives which were designed to be reconstituted into the core of LDL in place of the native cholesteryl esters. One of these derivates, cholesteryl 12–0-[methyl 3–0-methyl-5′(6′)-carboxyfluorescein]ricinoleyl carbonate (MMC) was reconstituted into the core of LDL. The resultant fluorescent reconstituted LDL was used in conjunction with flow cytometry to quantify the LDL receptor activity of fresh blood lymphocytes derived from normal individuals and from patients with the heterozygous and homozygous forms of familial hypercholesterolemia (FH). The LDL receptor activities of the heterozygous and homozygous FH lymphocytes were approximately 37% and 1% of normal, respectively. LDL reconstituted with these lipophilic fluorescein derivatives will be valuable in studying LDL metabolism and may be useful for the diagnosis of FH.     

Effect of bean intake on biliary lipid secretion and on hepatic cholesterol metabolism in the rat

We studied the effect of a bean diet on biliary lipid secretion, serum cholesterol concentration, and hepatic cholesterol metabolism in the rat. Rats fed a bean diet for 10-12 days had increased biliary cholesterol output and molar percentage by 300% and 200%, respectively, compared to rats fed an isocaloric and isoprotein casein diet. Biliary phospholipid output increased 180%. Bile flow and biliary bile salt output remained in the normal range. Total serum and VLDL cholesterol concentration significantly decreased 27% and 50%, respectively, in the rats fed the bean diet. Hepatic cholesterogenesis was increased 170% in the bean-fed animals. The relative contribution of newly synthesized hepatic cholesterol to total biliary cholesterol increased 200%, and that of endogenous origin only 50%. These results suggested that newly synthesized hepatic cholesterol was preferentially channelled to the biliary cholesterol secretory pathway in bean-fed rats. Although hepatic cholesteryl ester concentration increased 240%, the incorporation of [14C]oleate into hepatic cholesteryl esters was significantly decreased by 30% in isolated hepatocytes of bean-fed animals. These results were consistent with the possibility that the availability of hepatic free cholesterol for biliary secretion was increased in the bean-fed animals. This study demonstrates that bean intake has a profound effect on the metabolic channelling and compartmentalization of hepatic cholesterol, resulting in a significant decrease in total serum and very low density lipoprotein cholesterol concentrations and a high biliary cholesterol output.     

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.     

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.     

Intestinal HMG-CoA reductase activity is low in hypercholesterolemic patients and is further decreased with lovastatin therapy

Significant cholesterol synthesis occurs in gut mucosa of animals and humans. However, the role of gut synthesis in hypercholesterolemia and the effect of drugs on this function have not been defined. We obtained jejunal biopsies and bile samples from 21 Type II hypercholesterolemic subjects (mean serum cholesterol = 331 mg/dl) on a low fat diet after an over-night fast. Whole gut mucosal homogenate was assayed for activity of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the rate-determining enzyme of cholesterol synthesis. Mean reductase activity (pmol/mg per min) was 5.5 +/- 1.0 (n = 21) in hypercholesterolemic subjects versus 11.3 +/- 1.0 in 52 normal subjects (P less than 0.01). This is consistent with the hypothesis that the primary defect in these patients is not excessive cholesterol synthesis but decreased low density lipoprotein (LDL) clearance. It implies that high LDL levels down-regulate gut reductase activity. After treatment of 7 patients with lovastatin (40-80 mg/day for at least 6-13 weeks), gut reductase activity decreased from 7.7 +/- 2.6 to 3.6 +/- 0.5 (P less than 0.05), in biopsies obtained 12 hr after the last drug dose. Inhibition of reductase activity by this drug was detected 12 hr after a dose, and the enzyme was not measurably induced during 6-13 weeks of therapy. In keeping with the decrease in serum cholesterol (332----239 mg/dl) and mucosal reductase activity during lovastatin therapy, mean gallbladder bile cholesterol saturation index also decreased (1.045 +/- 0.112 vs. 0.883 +/- 0.109, n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased expression of LXR alpha, ABCG5, ABCG8, and SR-BI in the liver from normolipidemic, nonobese Chinese gallstone patients

Cholesterol supersaturation of bile is one prerequisite for gallstone formation. In the present study of Chinese patients with gallstones, we investigated whether this phenomenon was correlated with the hepatic expression of genes participating in the metabolism of cholesterol and bile acids. Twenty-two nonobese, normolipidemic patients (female-male, 11:11) with gallstones were investigated with 13 age- and body mass index-matched gallstone-free controls (female-male, 10:3). The bile from the gallstone patients had higher cholesterol saturation than that from the controls. The mRNA levels of ABCG5, ABCG8, and liver X receptor alpha (LXRalpha) in the gallstone patients were increased by 51, 59, and 102%, respectively, and significantly correlated with the molar percentage of biliary cholesterol and cholesterol saturation index (CSI). The mRNA and protein levels of the hepatic scavenger receptor class B type I (SR-BI) were increased, and a significant correlation was found between the protein levels and the CSI. No differences were recorded between the two groups concerning the hepatic synthesis of cholesterol, bile acids, and esterification of cholesterol. Our results suggest that the upregulation of ABCG5/ABCG8 in gallstone patients, possibly mediated by increased LXRalpha, may contribute to the cholesterol supersaturation of bile. Our data are consistent with the possibility that increased amounts of biliary cholesterol may originate from plasma HDL cholesterol by enhanced transfer via SR-BI.