Cholesterol Gallstone Susceptibility Research Articles

An analysis of the role of the indigenous microbiota in cholesterol gallstone pathogenesis.

Background and AimsCholesterol 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.MethodsGenetically 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.ResultsAlterations 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.ConclusionsThese 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.

The ABCG5 ABCG8 sterol transporter and phytosterols: implications for cardiometabolic disease

This review summarizes recent developments in the activity, regulation, and physiology of the ABCG5 ABCG8 (G5G8) transporter and the use of its xenobiotic substrates, phytosterols, as cholesterol lowering agents in the treatment of cardiovascular disease. Recent progress has significant implications for the role of G5G8 and its substrates in complications associated with features of the metabolic syndrome. Recent reports expand the clinical presentation of sitosterolemia to include platelet and adrenal dysfunction. The G5G8 sterol transporter is critical to hepatobiliary excretion of cholesterol under nonpathological conditions and has been linked to the cholesterol gallstone susceptibility. Finally, the cardiovascular benefits of cholesterol lowering through the use of phytosterol supplements were offset by vascular dysfunction, suggesting that alternative strategies to reduced cholesterol absorption offer greater benefit. Insulin resistance elevates G5G8 and increases susceptibility to cholesterol gallstones. However, this transporter is critical for the exclusion of phytosterols from the absorptive pathways in the intestine. Challenging the limits of this protective mechanism through phytosterol supplementation diminishes the cardioprotective benefits of cholesterol lowering in mouse models of cardiovascular disease.

Hepatic insulin resistance directly promotes formation of cholesterol gallstones

Despite the well-documented association between gallstones and the metabolic syndrome, the mechanistic links between these two disorders remain unknown. Here we show that mice solely with hepatic insulin resistance, created by liver-specific disruption of the insulin receptor (LIRKO mice) are markedly predisposed toward cholesterol gallstone formation due to at least two distinct mechanisms. Disinhibition of the forkhead transcription factor FoxO1, increases expression of the biliary cholesterol transporters Abcg5 and Abcg8, resulting in an increase in biliary cholesterol secretion. Hepatic insulin resistance also decreases expression of the bile acid synthetic enzymes, particularly Cyp7b1, and produces partial resistance to the farnesoid X receptor, leading to a lithogenic bile salt profile. As a result, after twelve weeks on a lithogenic diet, all of the LIRKO mice develop gallstones. Thus, hepatic insulin resistance provides a crucial link between the metabolic syndrome and increased cholesterol gallstone susceptibility.

Variation of the gene encoding the nuclear bile salt receptor FXR and gallstone susceptibility in mice and humans

From quantitative trait locus mapping in inbred mice, we identified the Nr1h4 gene encoding the nuclear bile salt receptor FXR as a candidate gene for the cholesterol gallstone susceptibility locus Lith7. Here, we investigated further an association of the gene encoding FXR and gallstone susceptibility in mice and humans. The Nr1h4 gene was sequenced in inbred mouse strains with susceptible and resistant Lith7 alleles. Quantitative RT-PCR was employed to determine mRNA expression levels. Gallstone carriers and control subjects of three different populations comprising 1004 individuals were genotyped for polymorphisms of the orthologous human gene detected by sequencing. Expression and sequence analyses in inbred mice were consistent with Nr1h4 underlying Lith7. In the human populations, we identified three frequent haplotypes that accounted for > 95% of all haplotypes observed. In a Mexican population, the most common haplotype NR1H4_1 was associated with gallstone prevalence. In contrast, NR1H4_1 displayed no association with gallstone prevalence in a German population, whereas in a Chilean population we observed a trend towards a protective effect of NR1H4_1. Our study in an inbred mouse model and in three ethnically distinct populations indicates complex interactions of NR1H4 alleles and other risk factors for the development of cholelithiasis.

T-Cell Function Is Critical for Murine Cholesterol Gallstone Formation

The formation of cholesterol gallstones is a complex process involving contributions from genes and environmental factors. Although gallbladder inflammation is believed to be common during cholelithogenesis, the role of immunologic factors is unknown. The role of adaptive immunity in cholesterol cholelithogenesis was analyzed utilizing immunocompetent Helicobacter spp.-infected and -uninfected BALB/c and congenic immunodeficient Rag2(-/-) (Rag) mice. Lymphocyte transfer studies were performed to determine which cellular subset was responsible for cholesterol gallstone formation. Also, gallbladder inflammation was quantified to determine the nature of the inflammatory response associated with cholelilithogenesis. When fed a lithogenic diet for 8 weeks, wild-type mice developed significantly more cholesterol gallstones (27%-80% prevalence) than Rag mice ( approximately 5%, P < .05). Helicobacter spp.-infected BALB/cJ mice displayed statistically significant increases in cholesterol gallstone prevalence compared with uninfected mice (81% vs. 39%; P < .05). Transfer of splenocytes or T lymphocytes to Rag2(-/-) mice increased stone prevalence markedly (26% and 40% respectively; P < .05), whereas transfer of B cells was not appreciably cholelithogenic (13%). The adaptive immune response increased the expression of gallbladder Muc genes and accumulation of mucin gel. In addition, T cells and cholesterol monohydrate crystals induced proinflammatory gene expression in the gallbladder, which likely contributes to gallbladder dysfunction. These studies indicate that T cells are critical in murine cholesterol cholelithogenesis. Furthermore, cholesterol monohydrate crystals induce expression of proinflammatory cytokines in a T-cell-dependent fashion. Acquired immunity and inflammation are likely to be crucial factors in cholesterol gallstone pathogenesis, rather then merely the result of cholelithogenesis.

Cholesterol Gallstone Susceptibility Loci: A Mouse Map, Candidate Gene Evaluation, and Guide to Human LITH Genes

Cholesterol gallstones are prevalent and costly, chiefly among developed countries. In addition to numerous environmental risk factors, a complex genetic basis determines the risk for developing cholesterol gallstones. Rather than monogenic mutations that are rare and occur in limited populations, genetic predisposition to gallstone susceptibility in general populations arises from polymorphisms in multiple genes, each making a small contribution to overall risk. Because mouse and human genomes are conserved and only a critical subset of homologous genes appears rate limiting for gallstone susceptibility in these species, a genetic map of mouse lithogenic ( Lith ) loci provides a roadmap for the discovery of human LITH genes. Quantitative trait locus (QTL) mapping was employed to identify Lith loci in mice. Repeated detection of colocalizing QTLs among 9 crosses of 12 genetically diverse progenitor strains suggests identification of most major Lith QTLs, including Lith1 – Lith23 . Therefore, using this knowledge, our priority is to predict human LITH genes. To date, predominantly, prior knowledge of gene-product function was invoked to postulate causal roles for genes in gallstone susceptibility. Unfortunately, few such genes colocalized with empirical Lith loci, suggesting absence of causality or very limited contributions. Consequently, we present a systematic and comprehensive analysis of literature to support or refute the contributions to gallstone susceptibility by genes located within critical regions of empirical QTLs. We envisage that identification of human LITH genes based on our translational approach will provide targets for the development of new means of prevention and nonsurgical management of cholelithiasis.

Association of a lithogenic Abcg5/Abcg8 allele on Chromosome 17 (Lith9) with cholesterol gallstone formation in PERA/EiJ mice

To examine further the genetic determinants of cholesterol gallstone susceptibility in inbred mice, we performed quantitative trait locus (QTL) analysis of an intercross of gallstone-susceptible PERA/EiJ and gallstone-resistant DBA/2J inbred mice. Three hundred twenty-four F2 offspring were phenotyped for cholelithiasis during consumption of a lithogenic diet and genotyped using microsatellite markers. Linkage analysis was performed by interval mapping. In addition, we analyzed the combined datasets from this cross and from an independent cross of strain PERA and gallstone-resistant I/Ln mice. QTL mapping detected one significant new gallstone susceptibility (Lith) locus on Chromosome 13 (Lith15). A second significant QTL on Chr 6 (Lith16) confirmed a previous QTL. Furthermore, suggestive QTLs confirmed Lith loci from previous crosses on Chromosomes 1, 2, 5, 16 and X. QTL analysis of the dataset derived from the combined crosses increased the detection power and narrowed confidence intervals of Lith loci on Chromosomes 2, 6, 13, and 16. Moreover, the analysis of combined datasets revealed a shared QTL between both crosses on Chromosome 17 (Lith9). Significantly higher mRNA expression of Abcg5 and Abcg8 in strain PERA compared with strains I/Ln and DBA/2 further substantiated that the PERA allele of Abcg5/Abcg8 was responsible for lithogenicity underlying Lith9.

Association of a Lithogenic Abcg5/Abcg8 Allele on Chromosome 17 (Lith9) with Cholesterol Gallstone Formation in PERA/EiJ Mice

We employ quantitative trait locus (QTL) mapping to identify genomic regions that harbor cholesterol gallstone susceptibility (Lith) genes in inbred mice. In a previous intercross of gallstone susceptible PERA and resistant I/LnJ mice, we detected a Lith locus on chromosome (Chr) 17 that was inherited from strain PERA and named Lith9. QTL mapping in an intercross of strain PERA with DBA/2J mice, another gallstone resistant strain, detected only a weak association of Lith9 with gallstone formation. We re-examined the genetic determinants of cholesterol gallstone susceptibility in strain PERA in a combined QTL analysis of the datasets from the two independent crosses sharing PERA as the gallstone susceptible parental strain. In addition, a haplotype analysis for the Lith9 region was conducted in the three parental strains based on single nucleotide polymorphisms and candidate genes were sequenced. Expression of candidate genes was determined by real-time PCR. QTL analysis of the dataset derived from the combined crosses confirmed a significant shared QTL in the Lith9 region with a gallstone susceptible allele in strain PERA and resistant alleles in strains I/Ln and DBA/2. The haplotype analysis of Lith9 identified a region of 3.4 megabase pairs with shared haplotypes between strains I/Ln and DBA/2 that differed from the haplotype of strain PERA. The narrowed Lith9 interval contained the candidate genes Abcg5 and Abcg8 encoding the canalicular cholesterol transporter. Sequencing confirmed identical Abcg5 and Abcg8 alleles in strains I/Ln and DBA/2 and different alleles of both genes in strain PERA. The PERA alleles of Abcg5 and Abcg8 were associated with significantly higher hepatic mRNA expression levels compared with strains I/Ln and DBA/2 that correlated positively with higher biliary cholesterol concentration. In conclusion, our analyses confirmed the Lith9 locus with a gallstone susceptible allele in strain PERA and narrowed the Lith9 interval. Higher expression levels of Abcg5/Abcg8 in strain PERA compared with I/Ln and DBA/2 substantiate the notion of dysregulated canalicular cholesterol transport as a principal cause of cholesterol gallstone formation in inbred mice.

Exploration of the Gene Encoding the Nuclear Bile Salt Receptor FXR as a Cholesterol Gallstone Susceptibility (LITH) Gene in a Mexican Population

Exploration of the Gene Encoding the Nuclear Bile Salt Receptor FXR as a Cholesterol Gallstone Susceptibility (LITH) Gene in a Mexican Population

Improved Power and Resolution of QTL Mapping for Cholesterol Gallstone Susceptibility Loci Based on Combined Analysis of Two Independent Crosses

Improved Power and Resolution of QTL Mapping for Cholesterol Gallstone Susceptibility Loci Based on Combined Analysis of Two Independent Crosses

FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait locus mapping in mice

Background & Aims : Cholesterol gallstone formation is a complex genetic trait. To identify additional cholesterol gallstone susceptibility loci, we performed a quantitative trait locus analysis using an intercross of PERA/Ei and I/LnJ inbred strains of mice. Methods : Mice of both sexes were examined for gallstone weight and evaluated according to a scoring system for the physical chemistry of cholelithiasis during feeding of a lithogenic diet. Intercross offspring were genotyped, and linkage analysis was performed by interval mapping. Differences in messenger RNA expression of positional candidate genes were determined using reverse-transcription and real-time polymerase chain reaction. Results : We identified significant loci associated with gallstone weight on chromosomes 10 and 4, named Lith7 and Lith8, respectively (both susceptibility alleles conferred by strain I/LnJ). Positional candidate genes with higher expression in I/LnJ mice are Fxr (official symbol, Nr1h4), encoding the nuclear bile salt receptor, on chromosome 10 and Shp1 (official symbol, Nr0b2), encoding the small heterodimer partner 1, on chromosome 4. A significant locus associated with gallstone score on chromosome 17, named Lith9 (susceptibility allele conferred by strain PERA/Ei), colocalizes with the genes Abcg5 and Abcg8 that encode the canalicular cholesterol transporter. Higher hepatic messenger RNA expression of Abcg5 and Abcg8 in strain PERA/Ei correlates positively with higher biliary cholesterol levels. Conclusions : Our findings suggest a primary role of the nuclear bile salt receptor FXR and the canalicular cholesterol transporter ABCG5/ABCG8 in the genetic susceptibility and pathogenesis of cholesterol cholelithiasis in these strains of inbred mice.

New quantitative trait loci that contribute to cholesterol gallstone formation detected in an intercross of CAST/Ei and 129S1/SvImJ inbred mice.

Cholesterol gallstone formation is a response to interactions between multiple genes and environmental stimuli. To determine the subset of cholesterol gallstone susceptibility (Lith) genes possessed by strains CAST/Ei (susceptible) and 129S1/SvImJ (resistant), we conducted quantitative trait locus (QTL) analyses of an intercross between these strains. Parental strains and F(1) mice of both genders were evaluated for gallstone formation after consumption of a lithogenic diet for 8 wk. Gallstone susceptibility of strain CAST was predominantly due to cholesterol hypersecretion. Male intercross offspring were genotyped and phenotyped for cholesterol gallstone formation after consumption of the lithogenic diet for 10 wk. Linkage analysis was performed using PSEUDOMARKER software. One significant, new QTL was detected and named Lith13 [chromosome (Chr) 5, 30 cM]. Statistical analyses and QTL fine mapping suggest this QTL may comprise two closely linked loci. We confirmed the presence of Lith6 (Chr 6). Suggestive QTL were detected on Chrs 1, 2, 5, 14, and 16. The QTL on Chrs 2 and 16 confirmed previously identified, suggestive QTL. Therefore, they were named Lith12 (101 cM) and Lith14 (42 cM), respectively. We identified candidate genes based on known function and location and performed mRNA expression analyses using both parental strains and intercross progeny for preliminary evaluation of their contributions to gallstone formation. Cebpb (Lith12), Pparg (Lith6), and Slc21a1 (Lith6) displayed expression differences. Our work continues to demonstrate the genetic complexity and to elucidate the pathophysiology of cholesterol gallstone formation. It should facilitate the development of new approaches for treating this common human disorder.

Mapping cholesterol gallstone susceptibility (Lith) genes in inbred mice.

The individual risk for developing cholesterol gallstones in response to specific environmental factors is determined by complex genetics involving multiple genes. In this review, we introduce inbred mice as a model to localise and identify the murine genes that harbour cholesterol gallstone susceptibility alleles (Lith genes). These genes are associated with increased risk of gallstone formation when mice are fed a lithogenic diet containing cholesterol and cholic acid. We summarise the steps involved in localising the chromosomal regions that harbour Lith genes, focusing particularly on the initial step known as quantitative trait locus mapping, which employs breeding crosses of gallstone-susceptible and gallstone-resistant inbred mouse strains. Subsequent steps to narrow the chromosomal regions of the quantitative trait loci and identify the underlying Lith genes are outlined, with particular reference to the examples of Lith1 and Lith2, the first discovered quantitative trait loci associated with murine cholesterol cholelithiasis. We have now reported five quantitative trait loci for murine cholelithogenesis, which are officially named Lith1 through Lith5. Once the genes underlying these quantitative trait loci and other chromosomal loci from ongoing mouse crosses are identified and confirmed, the 'road-map' for discovery of orthologous human LITH genes will be available and, thereafter, their putative roles in cholesterol gallstone formation can be tested in selected human populations.

The Abcc2-Lith2 locus induces hypersensitivity to the biliary lithogenic effect of oestrogens by precipitating the onset of lithogenic bile

Background and Aim: We recently established Abcc2 to be the molecular basis underlying Lith2, one of the major cholesterol gallstone susceptibility loci identified in inbred mice fed a lithogenic diet. Based on the direct role of ABCC2 in the hepatohiliary transport of different drug and hormone conjugates, many of which are well-recognised to constitute high risk factors for human cholelithiasis, we hypotbetised that the Lith2/Abcc2 locus impacts not only the fithogenic response to dietary intervention but also to drugs and hormones. We tested our hypothesis here, using estrogen as a model compound. Methods: AKi.L/th2 s congenic mice (Lith2) and AKR controls were injected i.p. with ethinyl estradiol (EE) at the dose of 1X or 3X (5 or 15mg/kg-day) for 3 days. Bile flow and biliary lipids were assessed from hepatic bile with standard procedures. Hepatic bile acids were assayed by LC-MS-MS. Results: Body weights were identical between groups, establishing that all mice received an equivalent amount of EE Without EE-treatment, biliary lipid secretion rates, biliary lipid coupling and CSI were undifferentiable between groups. After EE 1X, however, while the controls increased their bile salts (BS) secretion rate and modestly but significantly decreased their biliary secretion of phospholipids (PL) and cholesterol (Ch) (most likely due to the uncoupling effect of EE-conjugates), the Lith2 group exhibited a declining BS secretion rate, a drop 2 times more pronounced in PL secretion rates and a sharp rise in biliary Ch, culminating in a marked elevation of Ch/BS or PL ratios and CSI. Tripling the dose of EE had no additional effect in lath2 mice but led the controls to now develop an hepatobiliary and lithogenic profile undistinguishable from the congenic animals. Percent of hepatic dihydroxylated BS were elevated in all Lith2 mice treated with EE but only in the controls receiving EE 3X, thereby being in direct relationship with biliary lithogenicity. Conclusions: The Lith2 locus, and most likely Abcc2, is not solely involved in the high-lithogeinc risk following a lithogenic diet, but also induces an hypersensitivity to the hthogenic effect of estrogens. Dihydroxylated hepatic bile acids appears central to the development of lithogenic bile, possibly through activation of nuclear receptor/ABCG5-8 expression. We speculate that such gene-estrogen interaction plays a role in the lithogenic risk associated with gender, pregnancy and estrogen-based therapies.

473 Quantitative trait locus mapping in an intercross of PERA/EI and DBA/2J inbred mice: identification of new cholesterol gallstone susceptibility () loci and confirmation of loci from previous crosses

473 Quantitative trait locus mapping in an intercross of PERA/EI and DBA/2J inbred mice: identification of new cholesterol gallstone susceptibility () loci and confirmation of loci from previous crosses

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 (Ay), tubby (tub), leptin (Lepob), leptin receptor (Leprdb)] murine models of obesity during in-gestion 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 Lepob and the Leprdb 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 Cpefat 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.

Molecular genetics of cholesterol cholelithiasis: identification of human and murine gallstone genes

Cholesterol cholelithiasis is one of the most common gastroenterological diseases in Western countries. It is a polygenic disease resulting from disturbed biliary cholesterol homeostasis. Association studies identified six human gallstone candidate genes. Polymorphisms in the genes encoding the apolipoproteins B and E, phospholipid flippase ( ABCB4), cholesterol ester transfer protein ( CETP), cholesterol-7alpha-hydroxylase ( CYP7A1) and ileal bile acid transporter ( SLC10A2) are correlated with gallstone prevalence. Quantitative Trait Locus (QTL) analysis localises additional unknown gallstone genes in inbred mice. Based on the natural variation of cholesterol gallstone susceptibility among different inbred strains, 5 lithogenic ( Lith) loci have been identified. Hepatobiliary transporters (e. g. bile salt export pump Abcb11) and key proteins of the lipoprotein metabolism (e. g. hepatic lipase Lipc) could be established as creedal candidate genes for Lith loci. The rapid progress of mouse and human genome projects provides the basis for the analysis of orthologous human LITH genes in gallstone patients, which might offer new prospects for individual risk assessment and molecular targets for stone prevention.

Identification of 18 Mouse ABC Genes and Characterization of the ABC Superfamily in Mus musculus

ATP-binding cassette (ABC) genes encode a family of transport proteins known to be involved in a number of human genetic diseases. In this study, we characterized the ABC superfamily in Mus musculus through in silico gene identification and mapping and phylogenetic analysis of mouse and human ABC genes. By querying dbEST with amino acid sequences from the conserved ATP-binding domains, we identified and partially sequenced 18 new mouse ABC genes, bringing the total number of mouse ABC genes to 34. Twelve of the new ABC genes were mapped in the mouse genome to the X chromosome and to 10 of the 19 autosomes. Phylogenetic relationships of mouse and human ABC genes were examined with maximum parsimony and neighbor-joining analyses that demonstrated that mouse and human ABC orthologs are more closely related than are mouse paralogs. The mouse ABC genes could be grouped into the seven previously described human ABC subfamilies. Three mouse ABC genes mapped to regions implicated in cholesterol gallstone susceptibility.

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.

Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: integrated activities of hepatic lipid regulatory enzymes

There is no consensus whether hepatic lipid regulatory enzymes play primary or secondary roles in cholesterol cholelithiasis. We have used inbred mice with Lith genes that determine cholesterol gallstone susceptibility to evaluate the question. We studied activities of regulatory enzymes in cholesterol biosynthesis (HMG-CoA reductase), cholesterol esterification (acyl-CoA:cholesterol acyltransferase) and the "neutral" (cholesterol 7alpha-hydroxylase) and "acidic" (sterol 27-hydroxylase) pathways of bile salt synthesis in strains C57L/J and SWR/J as well as recombinant inbred (AKXL-29) mice, all of which have susceptible Lith alleles, and compared them to AKR/J mice with resistant Lith alleles. We determined hepatic enzyme activities of male mice before and at frequent intervals during feeding a lithogenic diet (15% dairy fat, 1% cholesterol, 0.5% cholic acid) for 12 weeks. Basal activities on chow show significant genetic variations for HMG-CoA reductase, sterol 27-hydroxylase, and acyl-CoA: cholesterol acyltranferase, but not for cholesterol 7alpha-hydroxylase. In response to the lithogenic diet, activities of the regulatory enzymes in the two bile salt synthetic pathways are coordinately down-regulated and correlate inversely with prevalence rates of cholesterol crystals and gallstones. Compared with gallstone-resistant mice, significantly higher HMG-CoA reductase activities together with lower activities of both bile salt synthetic enzymes are hallmarks of the enzymatic phenotype in mice with susceptible Lith alleles. The most parsimonious explanation for the multiple enzymatic alterations is that the primary Lith phenotype induces secondary events to increase availability of cholesterol to supply the sterol to the hepatocyte canalicular membrane for hypersecretion into bile.