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.     

Secretory phospholipase A2 increases SR-BI-mediated selective uptake from HDL but not biliary cholesterol secretion

High density lipoprotein cholesterol represents a major source of biliary cholesterol. Secretory phospholipase A2 (sPLA2) is an acute phase enzyme mediating decreased plasma HDL cholesterol levels. Clinical studies reported a link between increased sPLA2 expression and the presence of cholesterol gallstones. The aim of our study was to investigate whether the overexpression of human sPLA2 in transgenic mice affects biliary cholesterol secretion and gallstone formation. Liver weight (P < 0.01) and hepatic cholesterol content (P < 0.01) were significantly increased in sPLA2 transgenic mice compared with controls as a result of increased scavenger receptor class B type I (SR-BI)-mediated hepatic selective uptake of HDL cholesterol (P < 0.01), whereas hepatic SR-BI expression remained unchanged. However, biliary cholesterol secretion as well as fecal neutral sterol and fecal bile salt excretion remained unchanged in sPLA2 transgenic mice. Furthermore, gallstone prevalence in response to a lithogenic diet was identical in both groups. These data demonstrate that i) increased flux of cholesterol from HDL into the liver via SR-BI as a result of phospholipase modification of the HDL particle translates neither into increased biliary and fecal sterol output nor into increased gallstone formation, and ii) increased sPLA2 expression in patients with cholesterol gallstones might be a consequence rather than the underlying cause of the disease.     

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.     

Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology Of biliary lipid secretion.

The inbred C57L strain but not the AKR strain of mice carry Lith genes that determine cholesterol gallstone susceptibility. When C57L mice are fed a lithogenic diet containing 15% fat, 1% cholesterol, and 0.5% cholic acid, gallbladder bile displays rapid cholesterol supersaturation, mucin gel accumulation, increases in hydrophobic bile salts, and rapid phase separation of solid and liquid crystals, all of which contribute to the high cholesterol gallstone prevalence rates (D. Q-H. Wang, B. Paigen, and M. C. Carey. J. Lipid Res. 1997. 38: 1395;-1411). We have now determined the hepatic secretion rates of biliary lipids in fasting male and female C57L and AKR mice and the intercross (C57L x AKR)F(1) before and at frequent intervals during feeding the lithogenic diet for 56 days. Bile flow and biliary lipid secretion rates were measured in the first hour of an acute bile fistula and circulating bile salt pool sizes were determined by the "washout" technique after cholecystectomy. Compared with AKR mice, we found that i) C57L and F(1) mice on chow displayed significantly higher secretion rates of all biliary lipids, and larger bile salt pool sizes, as well as higher bile salt-dependent and bile salt-independent flow rates; ii) the lithogenic diet further increased biliary cholesterol and lecithin outputs, but bile salt outputs remained constant. Biliary coupling of cholesterol to lecithin increased approximately 30%, setting the biophysical conditions necessary for cholesterol phase separation in the gallbladder; and iii) no gender differences in lipid secretion rates were noted but male mice exhibited significantly more hydrophobic bile salt pools than females.We conclude that in gallstone-susceptible mice, Lith genes determine increased outputs of all biliary lipids but promote cholesterol hypersecretion disproportionately to lecithin and bile salt outputs thereby inducing lithogenic bile formation.     

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.     

Restoration of gallstone susceptibility by leptin in C57BL/6J ob/ob mice

The absence of leptin due to the ob mutation leads to obesity and confers resistance to diet-induced cholesterol gallstone formation in otherwise susceptible C57BL/6J mice. To investigate contributions of obesity and leptin to gallstone susceptibility, C57BL/6J ob/ob mice were treated daily with i.p. saline or recombinant murine leptin at low (1 microgram/g bw) or high (10 microgram/g bw) doses and were pair-fed a lithogenic diet (15% dairy fat, 1.25% cholesterol, 0.5% cholic acid). Weight loss in ob/ob mice increased in proportion to leptin dose, indicating that the lithogenic diet did not impair leptin sensitivity. In a dose-dependent manner, leptin promoted cholesterol crystallization and gallstone formation, which did not occur in saline-treated mice. Notwithstanding, leptin decreased biliary lipid secretion rates without enriching cholesterol in bile. Leptin did not affect bile salt hydrophobicity, but did increase the biliary content of the most abundant molecular species of phosphatidylcholine, 16:0-18:2. Treatment with leptin down-regulated 3-hydroxy-3-methylglutaryl CoA reductase and prevented cholesterol from accumulating in liver. Consistent with increased hepatic clearance, leptin decreased plasma HDL cholesterol concentrations. This was accommodated in liver without up-regulation of cholesterol 7alpha-hydroxylase or Acat. These data suggest that despite the lithogenic diet, endogenous sources constitute a significant proportion of biliary cholesterol during leptin-induced weight loss. Kinetic factors related to cholesterol nucleation, gallbladder contractility, or mucin secretion may have accounted for leptin-induced gallstone formation.     

Hepatic cholesterol transport from plasma into bile: implications for gallstone disease

PURPOSE OF REVIEW: The transhepatic traffic of cholesterol from plasma lipoproteins into the bile is critical for overall cholesterol homeostasis and its alterations may lead to cholesterol gallstone formation. This review summarizes recent progress in understanding the key hepatic cholesterol metabolism-related proteins and pathways that influence biliary secretion of cholesterol. RECENT FINDINGS: In cholesterol-fed apolipoprotein E knockout mice, the availability of dietary cholesterol for biliary disposal is decreased and diet-induced gallstone formation is impaired. Scavenger receptor class B type I is relevant for cholesterol transport from plasma HDL into the bile in chow-fed mice, however its expression is not critical for biliary cholesterol secretion and gallstone formation in lithogenic diet-fed mice. Intrahepatic cholesterol transport proteins (e.g. sterol carrier protein-2, Niemann Pick type C-1 protein) also determine liver cholesterol available for biliary secretion in mice. Genetic manipulation of canalicular ATP-binding cassette transporter G5 and G8 expression in mice has established their essential role for biliary cholesterol secretion. SUMMARY: Recent studies have underscored that different proteins involved in hepatic cholesterol transport regulate the availability of cholesterol for biliary secretion. These advances may provide new avenues for prevention and treatment of various disease conditions linked to abnormal cholesterol metabolism.     

Targeted disruption of the murine mucin gene 1 decreases susceptibility to cholesterol gallstone formation

Gallbladder mucins play a critical role in the pathogenesis of cholesterol gallstones because of their ability to bind biliary lipids and accelerate cholesterol crystallization. Mucin secretion and accumulation in the gallbladder is determined by multiple mucin genes. To study whether mucin gene 1 (Muc1) influences susceptibility to cholesterol cholelithiasis, we investigated male Muc1-deficient (Muc1(-/-)) and wild-type mice fed a lithogenic diet containing 1% cholesterol and 0.5% cholic acid for 56 days. Gene expression of the gallbladder Muc1 and Muc5ac was significantly reduced in Muc1(-/-) mice in response to the lithogenic diet. Muc3 and Muc4 levels were upregulated and were similar between Muc1(-/-) and wild-type mice. Little or no Muc2 and Muc5b mRNAs were detected. Muc1(-/-) mice displayed significant decreases in total mucin secretion and accumulation in the gallbladder as well as retardation of crystallization, growth, and agglomeration of cholesterol monohydrate crystals. At 56 days of feeding, gallstone prevalence was decreased by 40% in Muc1(-/-) mice. However, cholesterol saturation indices of gallbladder bile, hepatic secretion of biliary lipids, and gallbladder size were comparable in Muc1(-/-) and wild-type mice. We conclude that decreased gallstone formation in mice with disrupted Muc1 gene results from reduced mucin secretion and accumulation in the gallbladder.     

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.     

Leptin induces the hepatic high density lipoprotein receptor scavenger receptor B type I (SR-BI) but not cholesterol 7alpha-hydroxylase (Cyp7a1) in leptin-deficient (ob/ob) mice

Cholesterol elimination from the body involves reverse cholesterol transport from peripheral tissues in which the elimination of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by the liver and subsequent biliary excretion as free cholesterol and bile acids are important. In situations of peripheral fat and cholesterol accumulation, such as obesity, these pathways may be overloaded, contributing to increased cholesterol deposition. Leptin has an important role in obesity, suppressing food intake and increasing energy expenditure. This hormone, which is absent in genetically obese ob/ob mice, is also thought to be involved in the coordination of lipid excretion pathways, although available data are somewhat inconsistent. We therefore studied the expression of the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor as well as the rate-limiting enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase (Cyp7a1), in leptin-deficient ob/ob mice and their wild-type controls. In ob/ob mice, protein levels of both LDL receptor and SR-BI were reduced, whereas LDL receptor mRNA levels were increased and those of SR-BI were reduced, regardless of challenge with a 2% cholesterol diet. In ob/ob mice, the enzymatic activity and mRNA for Cyp7a1 were reduced, and the increase in response to dietary cholesterol was blunted. Upon short-term (2 days) treatment with leptin, a dose-dependent increase was seen in the SR-BI protein and mRNA, whereas the Cyp7a1 protein and mRNA were reduced. Our findings indicate that leptin is an important regulator of hepatic SR-BI expression and, thus, HDL cholesterol levels, whereas it does not stimulate Cyp7a1 and bile acid synthesis.     

Aging per se is an independent risk factor for cholesterol gallstone formation in gallstone susceptible mice

Cholesterol gallstones occur rarely in childhood and adolescence and increase linearly with age in both genders. To explore whether aging per se increases cholesterol saturation of bile and gallstone prevalence, and to investigate age-related changes in hepatic and biliary lipid metabolism, we studied gallstone-susceptible C57L mice and resistant AKR mice of both genders fed 8 weeks with a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butter fat starting at (young adult) 8, (older adult) 36, and (aged) 50-weeks-of-age. After the 8-week feeding, gallstone prevalence, gallbladder size, biliary lipid secretion rate, and HMG-CoA reductase activity were significantly greater but cholesterol 7alpha-hydroxylase activity was lower in C57L mice of both genders compared with AKR mice. Increasing age augmented biliary secretion and intestinal absorption of cholesterol, reduced hepatic synthesis and biliary secretion of bile salts, and decreased gallbladder contractility, all of which increased susceptibility to cholesterol cholelithiasis in C57L mice. We conclude that aging per se is an independent risk factor for cholesterol gallstone formation. Because aging increases significantly biliary cholesterol hypersecretion and gallstone prevalence in C57L mice carrying Lith genes, it is highly like that Longevity (aging) genes can enhance lithogenesis of Lith (gallstone) genes.     

Effect of beta-muricholic acid on the prevention and dissolution of cholesterol gallstones in C57L/J mice

This study investigated whether beta-muricholic acid, a natural trihydroxy hydrophilic bile acid of rodents, acts as a biliary cholesterol-desaturating agent to prevent cholesterol gallstones and if it facilitates the dissolution of gallstones compared with ursodeoxycholic acid (UDCA). For gallstone prevention study, gallstone-susceptible male C57L mice were fed 8 weeks with a lithogenic diet (2% cholesterol and 0.5% cholic acid) with or without 0.5% UDCA or beta-muricholic acid. For gallstone dissolution study, additional groups of mice that have formed gallstones were fed chow with or without 0.5% beta-muricholic acid or UDCA for 8 weeks. One hundred percent of mice fed the lithogenic diet formed cholesterol gallstones. Addition of beta-muricholic acid and UDCA decreased gallstone prevalence to 20% and 50% through significantly reducing biliary secretion rate, saturation index, and intestinal absorption of cholesterol, as well as inducing phase boundary shift and an enlarged Region E that prevented the transition of cholesterol from its liquid crystalline phase to solid crystals and stones. Eight weeks of beta-muricholic acid and UDCA administration produced complete gallstone dissolution rates of 100% and 60% compared with the chow (10%). We conclude that beta-muricholic acid is more effective than UDCA in treating or preventing diet-induced or experimental cholesterol gallstones in mice.     

Hepatic overexpression of murine Abcb11 increases hepatobiliary lipid secretion and reduces hepatic steatosis

Abcb11 encodes for the liver bile salt export pump, which is rate-limiting for hepatobiliary bile salt secretion. We employed transthyretin-Abcb11 and BAC-Abcb11 transgenes to develop mice overexpressing the bile salt export pump in the liver. The mice manifest increases in bile flow and biliary secretion of bile salts, phosphatidylcholine, and cholesterol. Hepatic gene expression of cholesterol 7alpha-hydroxylase and ileal expression of the apical sodium bile salt transporter are markedly reduced, whereas gene expression of targets of the nuclear bile salt receptor FXR (ileal lipid-binding protein, short heterodimer partner (SHP) is increased. Because these changes in gene expression are associated with an increased overall hydrophobicity of the bile salt pool and a 4-fold increase of the FXR ligand taurodeoxycholate, they reflect bile salt-mediated regulation of FXR and SHP target genes. Despite the increased biliary secretion of bile salts, fecal bile salt excretion is unchanged, suggestive of an enhanced enterohepatic cycling of bile salts. Abcb11 transgenic mice fed a lithogenic (high cholesterol/fat/cholic acid) diet display markedly reduced hepatic steatosis compared with wild-type controls. We conclude that mice overexpressing Abcb11 display an increase in biliary bile salt secretion and taurodeoxycholate content, which is associated with FXR/SHP-mediated changes in hepatic and ileal gene expression. Because these mice are resistant to hepatic lipid accumulation, regulation of Abcb11 may be important for the pathogenesis and treatment of steatohepatitis.     

The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism

The HDL receptor scavenger receptor class B type I (SR-BI), which mediates selective HDL cholesterol uptake, plays a role in murine HDL metabolism, reverse cholesterol transport and whole-body cholesterol homeostasis. SR-BI is found in the liver, where its expression is regulated by estrogen, dietary cholesterol and fat, and controls murine plasma HDL cholesterol levels and bile cholesterol secretion. SR-BI is also highly expressed in rodent steroidogenic cells, where it facilitates cholesterol uptake for storage or steroid hormone synthesis and where its expression is regulated by trophic hormones. The detailed mechanism(s) underlying SR-BI-mediated selective cholesterol uptake have not yet been elucidated. Further analysis of the molecular and cellular bases of SR-BI regulation and function should provide new insights into the physiology and pathophysiology of cholesterol metabolism.     

Hepatic scavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol and its transport into bile

The clearance of free cholesterol from plasma lipoproteins by tissues is of major quantitative importance, but it is not known whether this is passive or receptor-mediated. Based on our finding that scavenger receptor BI (SR-BI) promotes free cholesterol (FC) exchange between high density lipoprotein (HDL) and cells, we tested whether SR-BI would effect FC movement in vivo using [(14)C]FC- and [(3)H]cholesteryl ester (CE)-labeled HDL in mice with increased (SR-BI transgenic (Tg)) or decreased (SR-BI attenuated (att)) hepatic SR-BI expression. The initial clearance of HDL FC was increased in SR-BI Tg mice by 72% and decreased in SR-BI att mice by 53%, but was unchanged in apoA-I knockout mice compared with wild-type mice. Transfer of FC to non-HDL and esterification of FC were minor and could not explain differences. The hepatic uptake of FC was increased in SR-BI Tg mice by 34% and decreased in SR-BI att mice by 22%. CE clearance and uptake gave similar results, but with much slower rates. The uptake of HDL FC and CE by SR-BI Tg primary hepatocytes was increased by 2.2- and 2.6-fold (1-h incubation), respectively, compared with control hepatocytes. In SR-BI Tg mice, the initial biliary secretion of [(14)C]FC was markedly increased, whereas increased [(3)H]FC appeared after a slight delay. Thus, in the mouse, a major portion of the clearance of HDL FC from plasma is mediated by SR-BI.     

The Liver-Selective Thyromimetic T-0681 Influences Reverse Cholesterol Transport and Atherosclerosis Development in Mice

Background

Liver-selective thyromimetics have been reported to efficiently reduce plasma cholesterol through the hepatic induction of both, the low-density lipoprotein receptor (LDLr) and the high-density lipoprotein (HDL) receptor; the scavenger receptor class B type I (SR-BI). Here, we investigated the effect of the thyromimetic T-0681 on reverse cholesterol transport (RCT) and atherosclerosis, and studied the underlying mechanisms using different mouse models, including mice lacking LDLr, SR-BI, and apoE, as well as CETP transgenic mice.

Methodology/Principal Findings

T-0681 treatment promoted bile acid production and biliary sterol secretion consistently in the majority of the studied mouse models, which was associated with a marked reduction of plasma cholesterol. Using an assay of macrophage RCT in mice, we found T-0681 to significantly increase fecal excretion of macrophage-derived neutral and acidic sterols. No positive effect on RCT was found in CETP transgenic mice, most likely due to the observed decrease in plasma CETP mass. Studies in SR-BI KO and LDLr KO mice suggested hepatic LDLr to be necessary for the action of T-0681 on lipid metabolism, as the compound did not have any influence on plasma cholesterol levels in mice lacking this receptor. Finally, prolonged treatment with T-0681 reduced the development of atherosclerosis by 60% in apoE KOs on Western type diet. In contrast, at an earlier time-point T-0681 slightly increased small fatty streak lesions, in part due to an impaired macrophage cholesterol efflux capacity, when compared to controls.

Conclusions/Significance

The present results show that liver-selective thyromimetics can promote RCT and that such compounds may protect from atherosclerosis partly through induction of bile acid metabolism and biliary sterol secretion. On-going clinical trials will show whether selective thyromimetics do prevent atherosclerosis also in humans.     

The Syrian golden hamster strain LPN: a useful animal model for human cholelithiasis

The purpose of this study was to specify the main mechanisms at the origin of gallstone formation in very young (5-week old) or young adult (9-week old) LPN hamsters fed a sucrose-rich (normal lipid) lithogenic diet for one and four weeks, respectively. It was also to compare these mechanisms in the two strains of hamsters (LPN and Janvier) or when an anti-lithiasic diet was given by substituting 10% of the sucrose by beta cyclodextrin. The LPN strain of hamsters showed a very high incidence of cholesterol gallstones (73%) after receiving the lithogenic diet. The gallstone formation is very rapid and occurs in less than one week in very young hamsters which show a high cholesterol synthesis rate in the liver. The cholesterol and phospholipid concentrations in the bile, cholesterol saturation index (CSI) and hydrophobic index (HI) increased significantly, concomitantly with a higher liver cholesterol synthesis in very young hamsters and with a lower bile acid synthesis (neutral pathway: cholesterol 7alpha-hydroxylase, CYP7A1 and acidic pathway: sterol 27 hydroxylase, CYP27A1) in young adult hamsters. No significant changes in the lipoprotein receptor expression (LDLr, SR-BI) were observed after feeding the lithogenic diet. Adding ten per cent beta-cyclodextrin, a cyclic oligosaccharide that binds cholesterol and bile acids to the lithogenic diet at the expense of sucrose, induced a decrease in cholesterol bile secretion and in the CSI and HI and prevented cholesterol gallstone formation. Similarly, another strain of Syrian Golden hamsters (" Janvier ") which originally exhibited a smaller bile cholesterol concentration, lower liver cholesterol synthesis and higher CYP7A1/CYP27A1 activity ratio did not carry cholesterol gallstones when fed the lithogenic diet. The main parameters always found at the origin of cholelithiasis in the Hamster are discussed: a higher hepatic cholesterogenesis (HMGCoAR), a higher HMGCoAR/CYP7A1 activity ratio, a lower cholesterol ester storage capacity, a higher CYP27A1/CYP7A1 activity ratio correlated to a higher cholesterol secretion in the bile and higher CSI and HI. In LPN hamsters, the incidence of cholesterol gallstones is nil when CSI + HI < 0.8 and positive for CSI + HI > 0.9. An overall comparison of the data obtained in LPN Hamsters and in Man suggests that this hamster strain appears to be an interesting model for human cholelithiasis.     

Overexpression of estrogen receptor alpha increases hepatic cholesterogenesis, leading to biliary hypersecretion in mice

We explored whether there is an "estrogen-ERalpha-SREBP-2" (for estrogen-estrogen receptor subtype alpha-sterol-regulatory element binding protein-2) pathway for regulating hepatic cholesterol biosynthesis in ovariectomized AKR mice treated with 17beta-estradial (E2) at 6 microg/day or E2 plus the antiestrogenic agent ICI 182,780 at 125 microg/day and on chow or fed a high-cholesterol (1%) diet for 14 days. To monitor changes in cholesterol biosynthesis and newly synthesized cholesterol secreted into bile, incorporation into digitonin-precipitable sterols in mice treated with 25 mCi of [3H]water was measured in extracts of liver and extrahepatic organs 1 h later and in hepatic biles 6 h later. ERalpha upregulated SREBP-2, with resulting activation of SREBP-2-responsive genes in the cholesterol biosynthetic pathway. The E2-treated mice continued to synthesize cholesterol in spite of its excess availability from high dietary cholesterol, which reflects a loss in controlling the negative feedback regulation of cholesterol synthesis. These alterations augmented biliary cholesterol secretion and enhanced the lithogenicity of bile. However, these lithogenic effects of E2 were fully blocked by ICI 182,780. We conclude that during estrogen treatment, more newly synthesized cholesterol determined by the estrogen-ERalpha-SREBP-2 pathway is secreted into bile, leading to biliary cholesterol hypersecretion. These studies provide insights into therapeutic approaches to cholesterol gallstones in high-risk subjects, especially those exposed to high levels of estrogen.