High cholesterol absorption efficiency and rapid biliary secretion of chylomicron remnant cholesterol enhance cholelithogenesis in gallstone-susceptible mice

The study of chylomicron pathway through which it exerts its metabolic effects on biliary cholesterol secretion is crucial for understanding how high dietary cholesterol influences cholelithogenesis. We explored a relationship between cholesterol absorption efficiency and gallstone prevalence in 15 strains of inbred male mice and the metabolic fate of chylomicron and chylomicron remnant cholesterol in gallstone-susceptible C57L and gallstone-resistant AKR mice. Our results show a positive and significant (P<0.0001, r=0.87) correlation between percent cholesterol absorption and gallstone prevalence rates. Compared with AKR mice, C57L mice displayed significantly greater absorption of cholesterol from the small intestine, more rapid plasma clearance of chylomicrons and chylomicron remnants, higher activities of lipoprotein lipase and hepatic lipase, greater hepatic uptake of chylomicron remnants, and faster secretion of chylomicron remnant cholesterol from plasma into bile. All of these increased susceptibility to cholesterol gallstone formation in C57L mice. We conclude that genetic variations in cholesterol absorption efficiency are associated with cholesterol gallstone formation in inbred mice and cholesterol absorbed from the intestine provides an important source for biliary hypersecretion. Differential metabolism of the chylomicron remnant cholesterol between C57L and AKR mice clearly plays a crucial role in the formation of lithogenic bile and gallstones.     

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.     

Mechanism for the cholesterol-lowering action of egg white protein in rats

Eggs are a popular source of dietary cholesterol, but their consumption does not necessarily result in an increased serum cholesterol concentration. We investigated the cholesterol-lowering activity of egg white protein (EWP) and its potential mechanism in rats. The consumption of EWP resulted in a decreased concentration of cholesterol in the serum, liver and intestinal mucosa. The excretion of fecal neutral sterols and bile acids was greater by rats fed with EWP than by those fed with casein. The ratio of cholesterol and bile acids in the micellar phase to those in the solid phase was lower in the intestinal contents from rats fed with EWP than from those fed with casein. These results suggest that the cholesterol-lowering activity of EWP can be attributed to lowering the cholesterol absorption by intervening in the micellar formation in the intestines.     

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.     

Endurance exercise training reduces gallstone development in mice.

Gallstones form when the ratio of bile cholesterol to bile acids and phospholipids is elevated, causing cholesterol to precipitate. Physical inactivity is hypothesized to increase gallstone development, but experimental evidence supporting this is lacking, and potential mechanisms for the antilithogenic effects of exercise have not been described. The purpose of this study was to examine the effect of endurance exercise training on gallstone formation and the expression of genes involved in bile cholesterol metabolism in gallstone-sensitive (C57L/J) mice. At 10 wk, 50 male mice began a lithogenic diet and were randomly assigned to an exercise-training (EX) or sedentary (SED) group (n = 25 per group). Mice in the EX group ran on a treadmill at approximately 15 m/min for 45 min/day for 12 wk. At the time animals were euthanized, gallstones were collected, pooled by group, and weighed. The weight of the gallstones was 2.5-fold greater in the SED mice compared with EX mice (143 vs. 57 mg, respectively). In the EX mice, hepatic expression of the low-density lipoprotein receptor (LDLr), scavenger receptor class B type 1 (SRB1), and sterol 27 hydroxylase (Cyp27) was increased by approximately 2-fold (P < 0.05 for each). The LDLr and SRB1 increase cholesterol clearance by low-density lipoprotein and high-density lipoprotein particles, respectively, while Cyp27 promotes the catabolism of cholesterol to bile acids. Taken together, these data indicate that exercise promotes changes in hepatic gene expression that increase cholesterol uptake by the liver but simultaneously increase the catabolism of cholesterol to bile acids, effectively reducing cholesterol saturation in the bile. This suggests a mechanism by which exercise improves cholesterol clearance from the circulation while simultaneously inhibiting gallstone formation.     

Cholesterol, bile acid, and lipoprotein metabolism in two strains of hamster, one resistant, the other sensitive (LPN) to sucrose-induced cholelithiasis

A comprehensive study of cholesterol, bile acid, and lipoprotein metabolism was undertaken in two strains of hamster that differed markedly in their response to a sucrose-rich/low fat diet. Under basal conditions, hamsters from the LPN strain differed from Janvier hamsters by a lower cholesterolemia, a higher postprandial insulinemia, a more active cholesterogenesis in both liver [3- to 4-fold higher 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) activity and mRNA] and small intestine, and a lower hepatic acyl-coenzyme A:cholesterol acyltransferase activity. Cholesterol saturation indices in the gallbladder bile were similar for both strains, but the lipid concentration was 2-fold higher in LPN than in Janvier hamsters. LPN hamsters had a lower capacity to transform cholesterol into bile acids, shown by the smaller fraction of endogenous cholesterol converted into bile acids prior to fecal excretion (0.34 vs. 0.77). In LPN hamsters, the activities of cholesterol 7alpha-hydroxylase (C7OHase) and sterol 27-hydroxylase (S27OHase), the two rate-limiting enzymes of bile acid synthesis, were disproportionably lower (by 2-fold) to that of HMG-CoAR. When fed a sucrose-rich diet, plasma lipids increased, dietary cholesterol absorption improved, hepatic activities of HMG-CoA reductase, C7Ohase, and S27OHase were reduced, and intestinal S27OHase was inhibited in both strains. Despite a similar increase in the biliary hydrophobicity index due to the bile acid enrichment in chenodeoxycholic acid and derivatives, only LPN hamsters had an increased lithogenic index and developed cholesterol gallstones (75% incidence), whereas Janvier hamsters formed pigment gallstones (79% incidence).These studies indicate that LPN hamsters have a genetic predisposition to sucrose-induced cholesterol gallstone formation related to differences in cholesterol and bile acid metabolism.     

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.     

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.     

Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats

These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7alpha-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12alpha-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CB-groups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet.     

Identification of a Novel Hypocholesterolemic Protein,Major Royal Jelly Protein 1, Derived from Royal Jelly

Royal jelly (RJ) intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1), MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats.     

miRNA-223 Suppresses Mouse Gallstone Formation by Targeting Key Transporters in Hepatobiliary Cholesterol Secretion Pathway

miRNA-223 has been previously reported to play an essential role in hepatic cholesterol homeostasis. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. Hence, mice with conventional knockout (KO), hepatocyte-specific knockout (ΔHepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gall bladders and liver tissues were harvested for cholesterol crystal imaging, gallstone mass measurement and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. A 3'' UTR reporter gene assay was used to verify the direct target genes for miRNA-223. LD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, ΔHepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissues and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets expression. Therefore, the present study demonstrates a novel role of miRNA-223 in the gallstone formation by targeting ABCG5 and ABCG8 and elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease.     

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.     

Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol

Early responses of cholesterol metabolism to dietary cholesterol were compared between exogenous hypercholesterolemic (ExHC) and Sprague-Dawley rats. Both strains had a similar radioactivity of [¹⁴C]cholesterol in the serum half a day after the oral administration, but thereafter the radioactivity disappeared slowly in ExHC rats. ExHC rats promptly altered in response to the dietary cholesterol, activities of cholesterol 7α-hydroxylase and cholesterol synthesis in the liver and fecal excretion of bile acids derived from [¹⁴C]cholesterol administered orally. Lymphatic transport for 24 hr of [¹⁴C]cholesterol was similar between the strains. Triton administration resulted in a marked accumulation of cholesterol in serum d > 1.006 g/ml lipoproteins in ExHC rats; in addition, the formation of cholesteryl esters from [¹⁴C]oleic acid intravenously infused was greater in ExHC rats. These results indicate that ExHC rats increase serum cholesterol in response to exogenous cholesterol by decreasing the liver uptake and enhancing the secretion in the liver.     

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.     

Role of hydrophilic bile acids and of sterols on cholelithiasis in the hamster

The effect of various dietary additions such as cholesterol, beta-sitosterol, bile acids, and bile acid analogs on gallstone formation was studied in the hamster. Gallstones were formed in 50% of the animals fed a high glucose, fat-free diet. Administration of 0.2% cholesterol or 1% beta-sitosterol had no effect on the incidence of gallstones. Ursodeoxycholic acid (0.5%) and its analog ursodeoxy-oxazoline [2-(3 alpha, 7 beta-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] were ineffective in preventing gallstones. Hyodeoxycholic acid and hyodeoxy-oxazoline [2-(3 alpha,6 alpha-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] at the same dosage effectively prevented gallstones, while the trihydroxy bile acid, hyocholic acid, was not effective. Of all the dietary regimens tested, only hyodeoxycholic acid significantly lowered serum cholesterol. The lithogenic diet produced a five-fold increase in hepatic HMG-CoA reductase activity; this activity was not affected by dietary cholesterol or beta-sitosterol. Hyodeoxycholic acid and hyocholic acid feeding increased the reductase activity by an additional 50% while the other bile acids had no effect. beta-Sitosterol doubled the cholesterol 7 alpha-hydroxylase activity whereas hyodeoxy-oxazoline lowered it. Hyodeoxycholic acid-fed animals had significantly lower cholesterol absorption than the animals on the lithogenic diet alone. Biliary cholesterol content increased dramatically in the animals fed the lithogenic diet and was increased still further by ursodeoxycholic acid, hyodeoxycholic acid, and hyodeoxy-oxazoline. These data show that hyodeoxycholic acid and hyodeoxy-oxazoline do not prevent gallstones by inhibiting hepatic cholesterol synthesis or biliary cholesterol secretion.     

Prevention of cholesterol gallstone disease by FXR agonists in a mouse model

Cholesterol gallstone disease is characterized by several events, including cholesterol precipitation in bile, increased bile salt hydrophobicity and gallbladder inflammation. Here, we describe the same phenotype in mice lacking the bile acid receptor, FXR. Furthermore, in susceptible wild-type mice that recapitulate human cholesterol gallstone disease, treatment with a synthetic FXR agonist prevented sequelae of the disease. These effects were mediated by FXR-dependent increases in biliary bile salt and phospholipid concentrations, which restored cholesterol solubility and thereby prevented gallstone formation. Taken together, these results indicate that FXR is a promising therapeutic target for treating or preventing cholesterol gallstone disease.     

Cholesterol gallstone formation in overweight mice establishes that obesity per se is not linked directly to cholelithiasis risk

The relationship between obesity and cholesterol cholelithiasis is not well understood at physiologic or genetic levels. To clarify whether obesity per se leads to increased prevalence of cholelithiasis, we examined cholesterol gallstone susceptibility in three polygenic (KK/H1J, NON/LtJ, NOD/LtJ) and five monogenic [carboxypeptidase E (Cpe (fat)), agouti yellow (A(y)), tubby (tub), leptin (Lep(ob)), leptin receptor (Lepr (db))] murine models of obesity during ingestion of a lithogenic diet containing dairy fat, cholesterol, and cholic acid. At 8 weeks on the diet, one strain of polygenic obese mice was resistant whereas the others revealed low or intermediate prevalence rates of cholelithiasis. Monogenic obese mice showed distinct patterns with either high or low gallstone prevalence rates depending upon the mutation. Dysfunction of the leptin axis, as evidenced by the Lep(ob) and the Lepr (db) mutations, markedly reduced gallstone formation in a genetically susceptible background strain, indicating that in mice with this genetic background, physiologic leptin homeostasis is a requisite for cholesterol cholelithogenesis. In contrast, the Cpe (fat) mutation enhanced the prevalence of cholelithiasis markedly when compared with the background strain. Since CPE converts many prohormones to hormones, a deficiency of biologically active cholecystokinin is a likely contributor to enhanced susceptibility to cholelithiasis through compromising gallbladder contractility and small intestinal motility. Because some murine models of obesity increased, whereas others decreased cholesterol gallstone susceptibility, we establish that cholesterol cholelithiasis in mice is not simply a secondary consequence of obesity per se. Rather, specific genes and distinct pathophysiological pathways are responsible for the shared susceptibility to both of these common diseases.     

EP4 emerges as a novel regulator of bile acid synthesis and its activation protects against hypercholesterolemia

Prostaglandin E receptor subtype 4 (EP4) knockout mice develops spontaneous hypercholesterolemia but the detailed mechanisms by which EP4 affects cholesterol homeostasis remains unexplored. We sought to determine the cause of hypercholesterolemia in EP4 knockout mice, focusing on the role of EP4 in regulating the synthesis and elimination of cholesterol. Deficiency of EP4 significantly decreased total bile acid levels in the liver by 26.2% and the fecal bile acid content by 27.6% as compared to wild type littermates, indicating that the absence of EP4 decreased hepatic bile acid synthesis and their subsequent excretion in stools. EP4 deficiency negatively regulate bile acid synthesis through repression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK)-mediated cholesterol 7α-hydroxylase (CYP7A1) expression and that the hypercholesterolemia in EP4 knockout mice is due to a defect in cholesterol conversion into bile acids. Deficiency of EP4 also increased de novo cholesterol synthesis and altered cholesterol fluxes in and out of the liver. Treating high fat diet-challenged mice with the pharmacological EP4 agonist, CAY10580 (200?μg/kg body weight/day i.p) for three weeks effectively prevented diet-induced hypercholesterolemia, enhanced endogenous bile acid synthesis and their fecal excretion. In summary, EP4 plays a critical role in maintaining cholesterol homeostasis by regulating the synthesis and elimination of bile acids. Activation of EP4 serves as an effective novel strategy to promote cholesterol disposal in the forms of bile acids in order to lower plasma cholesterol levels.     

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.     

An Analysis of the Role of the Indigenous Microbiota in Cholesterol Gallstone Pathogenesis

Background and Aims

Cholesterol gallstone disease is a complex process involving both genetic and environmental variables. No information exists regarding what role if any the indigenous gastrointestinal microbiota may play in cholesterol gallstone pathogenesis and whether variations in the microbiota can alter cholesterol gallstone prevalence rates.

Methods

Genetically related substrains (BALB/cJ and BALB/cJBomTac) and (BALB/AnNTac and BALB/cByJ) of mice obtained from different vendors were compared for cholesterol gallstone prevalence after being fed a lithogenic diet for 8 weeks. The indigenous microbiome was altered in these substrains by oral gavage of fecal slurries as adults, by cross-fostering to mice with divergent flora at <1day of age or by rederiving into a germ-free state.

Results

Alterations in the indigenous microbiome altered significantly the accumulation of mucin gel and normalized gallbladder weight but did not alter cholesterol gallstone susceptibility in conventionally housed SPF mice. Germ-free rederivation rendered mice more susceptible to cholesterol gallstone formation. This susceptibility appeared to be largely due to alterations in gallbladder size and gallbladder wall inflammation. Colonization of germ-free mice with members of altered Schaedler flora normalized the gallstone phenotype to a level similar to conventionally housed mice.

Conclusions

These data demonstrate that alterations in the gastrointestinal microbiome may alter aspects of cholesterol gallstone pathogenesis and that in the appropriate circumstances these changes may impact cholesterol cholelithogenesis.