Effect of dietary fenugreek seeds on biliary proteins that influence nucleation of cholesterol crystals in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallising and anticrystallising factors present in bile. Dietary fenugreek seed has been recently observed to possess anti-lithogenic potential in experimental mice. In the current animal study, we evaluated the effect of dietary fenugreek on the compositional changes in the bile, particularly its effect on glycoproteins, low-molecular-weight (LMW) and high-molecular-weight (HMW) proteins, cholesterol nucleation time and cholesterol crystal growth. Groups of Wistar rats were fed for 10 weeks with diets: (1) basal control (C), (2) C + fenugreek (12%), (3) high cholesterol diet (HCD) and (4) HCD + fenugreek (12%). Feeding of HCD containing 0.5% cholesterol for 10 weeks rendered the bile lithogenic. Incorporation of fenugreek into HCD decreased the cholesterol content (70.5%), total protein (58.3%), glycoprotein (27.5%), lipid peroxides (13.6%) and cholesterol saturation index (from 1.98 to 0.75) in bile, increased the bile flow rate (19.5%), prolonged the cholesterol nucleation time and reduced the vesicular form of cholesterol (65%), which was accompanied with an increase in smaller vesicular form (94%). There was an increase in biliary phospholipid (33%) and total bile acid (49%) contents in the HCD + fenugreek group as compared with the HCD group. Electrophoretic separation of biliary LMW proteins showed the presence of a high concentration of 28-kDa protein, which might be responsible for the prolongation of cholesterol nucleation time in the fenugreek-fed groups. These findings indicate that the beneficial anti-lithogenic effect of dietary fenugreek, which primarily is due to reduction in the cholesterol content in bile, was additionally affected through a modulation of the nucleating and anti-nucleating proteins, which, in turn, affect cholesterol crystallisation.     

氨肽酶N的表达及其与结石形成的关系(英文)

 为研究大鼠高胆固醇饮食时 ,肝脏氨肽酶N(APN)在实验结石形成中可能的结石发生作用 ,采用 1.2 %胆固醇饮食 4周 ,诱发新西兰兔胆囊结石形成 .根据兔APN基因cDNA序列设计引物 ,提取肝脏总RNA .利用RT PCR检测肝脏APNmRNA水平的变化 ,用组织化学方法观察肝脏毛细胆管膜上APN的表达 .观察新西兰兔胆囊结石形成过程中肝脏APN的mRNA水平的变化、APN表达及胆汁中APN活性、胆脂、总蛋白含量的变化 ,探讨APN在胆石形成中可能的作用 .经成石饲料饲养后 ,随着胆汁饱和度增加和APN活性加强 ,胆囊结石组肝脏APNmRNA水平较对照组明显增高 ,胆囊结石组胆汁中总胆固醇、CSI、总蛋白浓度及APN活性均明显高于对照组 ,且胆汁中APN活性与肝脏APN的表达及胆汁CSI增高呈正相关 .结果提示 ,当存在胆汁过饱和的情况下 ,APN很可能作为促成核因子在胆结石形成早期发挥重要作用     

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.     

Characterization of the bile acid profile in developing male and female hamsters in response to dietary cholesterol challenge

The Syrian golden hamster is a frequently used model to study cholesterol and bile acid metabolism as well as cholesterol-induced cholelithiasis. However, diet-induced gallstones seem limited to young male hamsters of certain strains that develop depressed cholate/chenodeoxycholate bile acid ratios. To further elucidate gender and age specific aspects of cholesterol and bile acid metabolism, i.e. a possible age-related bile acid/gallstone relationship, plasma and biliary lipids and bile acid composition were analyzed in male and female hamsters under various physiological conditions of age and diet, the latter formulated with and without dietary cholesterol. During normal development (no cholesterol challenge) the percentage of cholic acid decreased while chenodeoxycholate increased, the shift being more pronounced in males. Furthermore, female hamsters had higher total plasma cholesterol than in males, while hepatic and biliary lipids did not differ. When challenged with excessive dietary cholesterol, female hamsters again developed significantly higher total plasma and hepatic cholesterol concentrations. Biliary lipids and cholesterol gallstone incidence revealed a significant gender effect with male hamsters developing a higher lithogenic index and more gallstones (cholesterol and pigment stones) than females. Female hamsters revealed a lower percentage of chenodeoxycholate and a higher percentage of cholate resulting in a more protective, higher cholate/cheno ratio (1.5 +/- 1.0) than in males (1.0 +/- 0.2). In summary, the bile acid pattern in developing and cholesterol-fed hamsters renders females less susceptible to gallstones, in part because they maintain more favorable biliary lipid and bile acid profiles, characterized by lower molar percentages of biliary cholesterol and chenodeoxycholate.     

Susceptibility to murine cholesterol gallstone formation is not affected by partial disruption of the HDL receptor SR-BI

High density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver where its cholesterol is secreted preferentially into bile. The scavenger receptor class B type I (SR-BI) is believed to play a pivotal role in unloading HDL cholesterol and its ester to hepatocytes. Here, using male SR-BI "att" mice with a dysfunctional mutation in the Sr-b1 promoter, we studied whether approximately 50% of normal SR-BI expression influences gallstone susceptibility in these mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid and 15% butterfat. Our results showed that the disruption of SR-BI expression reduced cholesterol secretion by 37% in the chow-fed state and 10% on the lithogenic diet, and while delaying incidence slightly, did not influence cumulative susceptibility to cholesterol gallstones. The lithogenic diet induced marked increases in biliary cholesterol and phospholipid secretion rates but not of bile salts. Basal expression of hepatic SR-BI protein was dissimilar in both wild-type and SR-BI mice, and remained unaltered in response to the lithogenic diet. By two independent dual isotope methods, intestinal cholesterol absorption was unimpaired by attenuation of the SR-BI which also displays low-density expression on small intestinal enterocytes. We conclude that although HDL cholesterol is a principal source of biliary cholesterol in the basal state, uptake of cholesterol from chylomicron remnants appears to be the major contributor to biliary cholesterol hypersecretion during diet-induced cholelithogenesis in the mouse.     

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.     

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.     

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.     

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.     

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.     

Effects of a Daisaikoto Extract on the Biliary Constituents in Mice and Rats

The effects of an extract of Daisaikoto (a traditional Chinese medicine) on biliary constituents was studied in mice fed with a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate (the control diet) and in rats fed with a cholesterol-free diet. The Daisaikoto extract was added to the control diet at a level of 0.5%. A high incidence of cholesterol gallstones were found in the control mice, but not in the mice given the Daisaikoto extract. This difference was concluded to have been due to the absolute concentration of bile acid in the bile being significantly higher in the mice given the Daisaikoto extract than in the control mice. The result from rats fed with the cholesterol-free diet also demonstrated that the Daisaikoto extract caused an increase in the absolute concentration of bile acid in the bile.     

Alterations in biliary lipids of mice during dehydrocholic acid feeding

Mice were fed a lithogenic diet consisting of Purina chow and 0.5% dehydrocholic acid (DHA group). Controls received Purina chow. Every 2 wk for 20 wk animals were killed, and biliary phospholipid, cholesterol, and bile salt concentrations were determined, as well as the extent of gallstone formation. With time there was a gradual, significant decline in the concentration and the relative composition of phospholipid in both groups compared with initial values. There was a significant increase in biliary cholesterol concentration and relative amount in the DHA group compared with the control. No significant differences were found in the relative amounts of bile salt or phospholipid between the two groups. Feeding DHA resulted in an increased concentration of bile salts and the sum of measured lipid compared with controls. After 8 wk, gallstones were found in approximately 60% of autopsied animals and correlated with increased cholesterol concentration. Our data support the hypothesis that there is a component of cholesterol secretion that may not be bile salt- or phospholipid-dependent. Our data also suggest that biliary phospholipid secretion decreases with age.     

成石前后胆囊粘膜上皮多聚核蛋白体RNA含量变化—胆液糖蛋白和蛋白质增加的机制探讨

胆囊淤泥是胆囊结石的前身。测定胆囊粘膜上皮游离(FPR)和结合多聚核蛋白体(MBPR)的RNA含量及胆液粘液糖蛋白(MGP)的结果表明:胆囊淤泥患者胆囊粘膜合成和分泌MGP明显亢进,MBPR与相应GB中MGP的相关性比较提示此时尚有胆液淤积存在,且FPR的RNA含量也显著增高,提示细胞增殖加快。荧光胺法测定胆液蛋白质的含量发现,胆囊淤泥和胆固醇性结石患者胆囊液GB蛋白质含量显著高于色素性结石患者及“正常”对照,但蛋白质的GB浓度与HB浓度的比值显著低于胆固醇GB/HB比值,且GB中MGP与蛋白质含量呈显著正相关。说明蛋白质含量的增加与GB中过高的MGP阻止胆囊中蛋白质的清除过程有关。成石前GB中MGP和蛋白质含量的同时增高及其机制的确立,对认识及预防体内结石均有意义。     

Immunoglobulins as nucleating proteins in the gallbladder bile of patients with cholesterol gallstones.

The gallbladder bile of patients with cholesterol gallstones contains pronucleating proteins which accelerate precipitation of cholesterol crystals from bile. In this study we have improved the purification procedure developed earlier for these nucleating proteins and have now identified the nature of these proteins. Gallbladder bile from patients with cholesterol gallstones was applied to concanavalin A affinity columns. The ConA-binding glycoprotein fractions containing the nucleating proteins were then separated by FPLC (fast protein liquid chromatography) using a Superose 12 gel filtration column. Nucleating activity was detected in the high molecular weight (FPLC-1) as well as in the low molecular weight fractions (FPLC-3). Investigation of the high molecular weight fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution and amino acid sequencing suggested that these proteins were immunoglobulins. Immunostaining of Western blots with specific monoclonal antibodies identified the presence of immunoglobulin (Ig) M and IgA in the FPLC-1 fraction. These immunoglobulins were further purified by affinity chromatography employing an antibody exchanger (ABx) column which specifically binds immunoglobulins. There was no reduction in the cholesterol nucleating activity in the Abx-bound fraction compared to FPLC-1. Additional studies showed that the FPLC-1 fraction was significantly more potent than the ConA glycoproteins from either rapid and slow nucleating biles. Also the number of crystals formed was significantly greater in the FPLC-1 fraction isolated from cholesterol gallstone biles than from the FPLC-1 fraction from control patient biles. Commercially obtained IgM and IgA had no effect on nucleation, but IgM isolated from the serum of patients with Waldenstrom's macroglobulinemia did accelerate the nucleation of cholesterol. We conclude that the IgM and possibly IgA are pronucleating proteins and may be important in the pathogenesis of cholesterol gallstones in man.     

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.     

Phosphatidylcholine-enriched diet prevents gallstone formation in mice susceptible to cholelithiasis

Cholesterol gallstones affect approximately 10-15% of the adult population in North America. Phosphatidylcholine (PC) is considered to be the main cholesterol solubilizer in bile. This study examined the effect of a PC-enriched diet on gallstone incidence in mice susceptible to cholelithiasis. The result obtained showed that the feeding of a lithogenic (LG) diet for 4 weeks or 8 weeks resulted in cholesterol gallstone incidences of 47% and 89%, respectively. These gallstone incidences were either reduced or prevented when the LG diet was enriched with 2% or 6% PC, respectively. The cholesterol saturation index (CSI) was reduced only in mice fed with LG + 6% PC diet as compared with mice fed the LG diet alone. However, in all groups, the CSI was significantly higher than in mice fed Purina chow diet. The biliary anionic polypeptide fraction (APF) was significantly increased in mice fed the LG + 2% PC diet and was reduced in those fed with LG + 6% PC diet. In conclusion, prevention or delay of gallstone formation was not due to a consistent effect on biliary lipid composition, suggesting a direct effect of PC on cholesterol solubilization and/or the effect of an additional nonlipid biliary component such as APF.     

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.     

Mice overexpressing hepatic Abcb11 rapidly develop cholesterol gallstones

Cholelithiasis is a polygenic disease, although the genes responsible for gallstone formation have not yet been clearly identified. QTL analysis has identified the Lith 1 loci on mouse Chromosome 2, and the hepatic bile salt transporter Abcb11 maps to the Lith 1 locus. We have used recently developed TTR-Abcb11 transgenic mice that overexpress Abcb11 to determine the effects of Abcb11 overexpression on cholesterol gallstone formation. TTR-Abcb11 and FVB/NJ strain control mice were fed a lithogenic or chow diet and cholesterol crystal and gallstone formation were measured. Biliary lipids in gallbladder bile and gene expression of canalicular lipid transporters were also analyzed. TTR-Abcb11 mice fed a lithogenic diet had an increased rate of cholesterol crystal and gallstone formation. This was associated with an increase in both the hydrophobic bile salt and cholesterol content of gallbladder bile. Expression of Abcb4, Abcg5, and Abcg8 did not change before gallstone formation. These data indicate that hepatic overexpression of Abcb11 increases the rate of cholesterol gallstone formation. This is likely because of increases in bile salt hydrophobicity but not because of alterations of other biliary lipid transporters. These findings strongly support Abcb11 as a Lith 1 gene.     

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.