FXR dysregulation as the metabolic nexus driving cholesterol gallstone disease.

Cholesterol gallstone disease is one of the most prevalent and the most costly digestive diseases in Western countries. Its pathogenesis is a complex paradigm resulting from the interaction of genetic factors, hepatic hypersecretion of cholesterol, increased intestinal absorption of cholesterol, a constantly "supersaturated" bile, crystallization of biliary cholesterol, and gallbladder stasis. De novo cholesterol biosynthesis, biliary cholesterol output, and intestinal cholesterol absorption are therefore key steps involved in cholesterol homeostasis. Establishing the right pharmacological therapy for cholesterol gallstones is of major importance in Western healthcare systems. Certain drugs might independently influence cholesterol gallstone formation by blocking the 3-hydroxy-3-methylglutaryl-coenzyme A reductase and inhibiting cholesterol biosynthesis in the liver (statins) or blocking cholesterol absorption in the small intestine apical membrane by specifically inhibiting the Niemann-Pick C1-like 1 protein (ezetimibe). This review will focus on the possibility that statins and ezetimibe, by acting at different levels of cholesterol homeostasis, might represent novel therapeutic approaches to prevent cholesterol gallstones in selected subjects at risk.

Cholesterol gallstone disease is a common condition in western populations. The etiology is multifactorial with interaction of genetic and environmental factors. Obesity, aging, estrogen treatment, pregnancy and diabetes are consistently associated to a higher risk. A number of dietary factors have been involved in the pathogenesis of cholelithiasis. In this article we summarize several studies that have evaluated the role of diet as a potential risk factor for gallstone formation, including energy intake, cholesterol, fatty acids, fiber, carbohydrates, vitamins and minerals, and alcohol intake. Consumption of simple sugars and saturated fat has been mostly associated to a higher risk, while fiber intake and moderate consumption of alcohol, consistently reduce the risk. The association between cholesterol intake and gallstone disease has been variable in different studies. The effects of other dietary factors are less conclusive; additional studies are therefore necessary to clarify their relevance in the pathogenesis of gallstone disease. Recent discoveries of the role of orphan nuclear receptors in the regulation of fatty acid and hepatic cholesterol metabolism and excretion open new perspectives for a better understanding of the role of dietary constituents on cholesterol gallstone formation.

Objective To evaluate if expression of serum retinol-binding protein 4 (RBP4) concentrations in cholesterol gallstone disease.Methods Serum RBP4 levels of 100 cholesterol gallstone disease patients (cholesterol gallstone disease group) and 147 healthy controls (healthy control group) were measured by enzyme-linked immunosorbent assay and further correlated with clinical and biochemical characteristics,including insulin resistance and renal function.The chemical composition of gallstones was determined by postoperative pathology.Results The level of RBP4 in cholesterol gallstone disease group was significantly lower than that in healthy control group [(30.57 ± 13.64 ) mg/L vs.(41.52 ± 20.25 ) mg/L](P＜ 0.01 ).The level of RBP4 was also associated with gallstone occurrence (OR =0.93,95％ CI:0.88 -0.96,P =0.004).Serum RBP4 levels of all subjects were positively correlated with total cholesterol,triglyceride,creatinine,insulin resistance and albumin ( P ＜ 0.05 or ＜ 0.01 ),and negatively correlated with aspartate aminotransferase (P ＜0.01).In multivariate analysis,cholesterol gallstone formation was significantly associated with a lower serum RBP4 level (OR =2.97,95％ CI:1.15 -7.68,P =0.025 ).Both gallstone patients and controls were subdivided into two groups according to ceatinine:≥ 88.40 μ mol/L group and ＜ 88.40 μ mol/L group.Patients with gallstones were found to have significantly lower serum RBP4 levels than controls in both subgroups (P =0.012,0.045 ).According to GFR,both gallstone patients and controls were subdivided into ≥ 90 ml/(min· 1.73 m2) group,60 - 89 ml/(min· 1.73 m2) group and ＜60 ml/ (min· 1.73 m2) group.It showed that a lower GFR was associated with greater serum RBP4 level in healthy control group.This trend was not noted in cholesterol gallstone disease group.Conclusions Serum RBP4 decreases in cholesterol gallstone disease independent of renal function.The relationship between liver function and RBP4 level in these patients deserves further investigation. Key words: Calculi; Cholesterol; Retinol-binding proteins

Cholesterol gallstone disease is initiated in a liver which produces abnormal bile with excess cholesterol relative to bile salts and phospholipid. To define the responsible secretory mechanism(s), the rate of biliary lipid secretion was measured by a duodenal marker perfusion technique, while the bile salt pool was simultaneously estimated by isotope dilution. Two groups of control patients expected to have normal biliary lipid composition--14 subjects without hepatobiliary disease and 6 patients with pigment gallstones, were compared to two experimental groups expected to have abnormal bile--10 nonobese patients with cholesterol gallstones and 7 obese subjects without gallstones. Both control groups had nearly identical biliary lipid secretion rates, and a corresponding low relative molar concentration of cholesterol. Two different secretory mechanisms were found to be responsible for the abnormal bile in the experimental groups. In the nonobese patients with cholesterol gallstones, bile salt and phospholipid secretion rates were both significantly reduced. Conversely, the grossly obese subjects had an increased cholesterol secretion. To determine how cholecystectomy improves biliary lipid composition, three groups of gallstone patients --6 with pigment stones, 4 grossly obese with cholesterol stones, and 13 nonobese with cholesterol stones --were all examined after full recovery from surgery. In the nonobese patients with cholesterol gallstones, both bile salt and phospholipid secretion significantly increased, causing a definite improvement in bile composition. Cholecystectomy produced a similar but less marked trend in the obese patients with cholesterol stones, and in the patients with pigment stones. Cholesterol secretion, however, was unaffected by surgery. The bile salt pool was definitely small in the nonobese patients with cholesterol gallstones and became slightly smaller after cholecystectomy. The pool was significantly reduced by cholecystectomyin the patients with cholesterol gallstones. Removal of the gallbladder in all three groups caused a greater fraction of the pool to cycle around the enterohepatic circulation each hour. This more rapid cycling produced the increase in bile salt and phospolipid secretion, and was responsible for the improved composition found after cholecystectomy.

Apolipoprotein E (apoE) isoforms are genetic determinants of interindividual variations in lipid metabolism. To assess whether apoE is a genetic risk factor for cholesterol gallstone disease (GD), we analyzed apoE variants in populations from Chile and Germany, two countries with very high prevalence rates of this disease. ApoE genotypes were determined in Chilean gallstone patients (n = 117) and control subjects (n = 122) as well as in German gallstone patients (n = 184) and matched controls (n = 184). In addition, we studied apoE variants in subgroups of Chilean patients with strong differences in their susceptibility to acquire gallstones: 50 elderly subjects without gallstones in spite of well-known risk factors for this disease (gallstone-resistant) and 32 young individuals with gallstones but without risk factors (gallstone-susceptible). Furthermore, correlation analysis of apoE genotypes with cholesterol crystal formation times, biliary cholesterol saturation index (CSI), and gallstone cholesterol contents was performed in 81 cholecystectomized patients. In this study analyzing the largest sample set available, apoE4 genotype was not associated with an increased frequency of GD in either population. Moreover, in the Chilean population after adjusting for risk factors such as gender, age, body mass index, serum lipids, and glucose, the odds ratio for the association of the apoE4 allele and GD was significantly (P < 0.05) <1. Also, genotypes were not correlated with cholesterol crystal formation time, CSI, or gallstone cholesterol content. In contrast to previous smaller studies, apoE polymorphisms were not associated with susceptibility to cholesterol GD in high-risk populations.

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.

This article presents an up to date of selected aspects of the molecular mechanisms of cholesterol metabolism most likely involved in cholesterol gallstones disease, a highly prevalent disease in the Western world. The etiology of cholesterol cholelithiasis is considered to be multifactorial, with interaction of genetic and environmental factors. The production of supersaturated bile by the liver of cholesterol is a key early metabolic event underlying cholesterol lithogenesis. Regulation of hepatic cholesterol trafficking within the hepatocyte appears essential for the production of cholesterol supersaturated bile. Impaired sorting of metabolically active hepatic free cholesterol to the bile acid biosynthetic or lipoprotein production pathways leads to an increased availability of cholesterol for preferential channeling of cholesterol to the canalicular membrane and further secretion into bile. Many of these intrahepatic cholesterol trafficking steps are under genetic control and might be influenced by a variety of environmental factors. This review summarizes recent discoveries related to transhepatic cholesterol flux and biliary lipid secretion, which have provided new insights to the regulation of hepatic cholesterol metabolism as related to gallstone disease.

Dietary fish oil inhibits cholesterol monohydrate crystal nucleation and gallstone formation in the prairie dog

Cholesterol gallstone disease (CGD) is one of the most common digestive diseases, and it is closely associated with hepatic cholesterol metabolism. Cholesterol gallstones may be caused by abnormal hepatic cholesterol metabolism, such as excessive cholesterol biosynthesis within the liver, interfering with the uptake or export of cholesterol in the liver, and abnormal hepatic cholesterol esterification. In this review, we begin with a brief overview of the clinical diagnosis and treatment of gallstone disease (GSD). Then, we briefly describe the major processes of hepatic cholesterol metabolism and summarize the key molecular expression changes of hepatic cholesterol metabolism in patients with gallstones. We review and analyze the recent advances in elucidating the relationships between these key molecules and CGD, and some targets significantly impacting on CGD via hepatic cholesterol metabolism are also listed. We also provide a significant discussion on the relationship between CGD and nonalcoholic fatty liver disease (NAFLD). Finally, the new discoveries of some therapeutic strategies associated with hepatic cholesterol metabolism to prevent and treat CGD are summarized.

Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice

The objective of this study was to investigate cholesterol metabolism in human gallbladder mucosa, especially in relation to hepatic cholesterol metabolism, gallstone disease and treatment with bile acids. Gallbladder mucosa and liver tissue samples were collected in 44 patients undergoing cholecystectomy; 30 had cholesterol gallstones and the rest were stone free. Ten of the gallstone patients were treated with chenodeoxycholic acid and eight received ursodeoxycholic acid, with a daily dose of 15 mg/kg body wt, for 3 wk before surgery. The 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, governing cholesterol synthesis, was considerably lower in the gallbladder mucosa than in liver tissue (28 +/- 6 and 120 +/- 40 pmol/min/mg protein). The acyl coenzyme A:acyltransferase activity in the gallbladder mucosa catalyzing the esterification of cholesterol was, on the other hand, several times higher than corresponding activity in the liver (92 +/- 23 and 11 +/- 2 pmol/min/mg protein). In the presence of exogenous cholesterol, the acyl coenzyme A:acyltransferase activity increased about twofold in the gallbladder mucosa. The acyl coenzyme A:acyltransferase activity of the gallbladder mucosa from untreated gallstone patients was not stimulated further by the addition of exogenous cholesterol. Otherwise, there were no significant differences in acyl coenzyme A:acyltransferase and 3-hydroxy-3-methylglutaryl coenzyme A reductase activities in the gallbladder mucosa of gallstone patients compared with gallstone-free controls. Treatment with chenodeoxycholic and ursodeoxycholic acids did not affect the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity of the gallbladder mucosa but reduced the acyl coenzyme A:acyltransferase activity by 60% to 65%.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of biliary cholesterol secretion by hepatocytes has received much interest [1], since it has a major impact on two highly relevant disease complexes, namely, the generation of cholesterol gallstones which have an overall prevalence of 10–20 % in developed countries [2] and reverse cholesterol transport [3], an important protective pathway against atherosclerotic cardiovascular disease as well as the main cause for morbidity and mortality in industrialized societies [4]. Less attention has thus far been given to the potential participation of the gallbladder itself in metabolism, which is somewhat surprising, since the gallbladder has the capacity to actively absorb cholesterol from bile and thereby modulate bile cholesterol content [5]. Changes in transporters mediating this metabolic activity of gallbladder epithelial cells could conceivably contribute to cholesterol gallstone formation in disease. However, only a very limited number of studies exist that have functionally explored the relative contributions of known cholesterol transport proteins to cholesterol transport in and out of the gallbladder (Fig. 1). The study by Yoon et al. [6] in this issue of Digestive Diseases and Sciences reports that the ATP-binding cassette transporters G5/G8 (ABCG5/G8) are expressed on the apical side, while ABCA1 is found on the basolateral side of gallbladder epithelial cells. Both transporters are upregulated in gallbladders from patients with cholesterol gallstone disease [6]. Thus far, on the apical side the expression of ACBG5/G8 [7], scavenger receptor class B type I (SR-BI) [8], cubilin and megalin [9] was reported in gallbladder epithelial cells. The obligate heterodimer ABCG5/G8 is a cholesterol transporter expressed in enterocytes and hepatocytes, where it mediates the excretion of cholesterol and plant sterols out of the cell [1]. About 70 % of total biliary cholesterol secretion is ABCG5/G8 dependent. Total genetic deletion of ABCG5/ G8 produces sitosterolemia, a disease associated with hyperabsorption of cholesterol and plant sterols that is linked to accelerated atherosclerosis [1]. Interestingly, the ABCG5/G8 locus has also long been implicated in the formation of cholesterol gallstones [10]. Recently, specific coding variants have been identified in genome-wide association studies that likely result in a gain-of-function of ABCG5/G8, supposedly translating into increased biliary cholesterol secretion [11]. Neither in animal experiments nor in human material has the functionality of ABCG5/G8, specifically in the gallbladder, been explored; although, derived from their function in liver and intestine increasing cholesterol secretion into bile would be expected. In this context the higher expression of these transporters noted by Yoon et al. [6] in patients with gallstone disease points towards a causative contribution of ABCG5/G8 to gallstone formation. However, results from studies using the perfused gallbladder model indicated reduced cholesterol absorption from bile in gallbladders from gallstone disease patients [12], although the methods used did not formally distinguish between decreased uptake versus uptake followed by accelerated resecretion, e.g. via increased ABCG5/G8 expression [12]. Moreover, decreased expression of uptake receptors on the apical side such as cubilin or megalin might also explain these results. Interestingly, cholesterol uptake and secretion can occur on the apical side [13] providing further indirect evidence for a potential functional role of ABCG5/G8 in the gallbladder. A. Dikkers U. J. F. Tietge (&) Department of Pediatrics, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands e-mail: u_tietge@yahoo.com

Biliary Lipid Output and Bile Acid Kinetics in Cholesterol Gallstone Disease

The apolipoprotein (apo) B gene Xba I polymorphism is associated with alterations in serum lipids. Disturbances in serum lipids may be a risk factor for cholesterol gallstone disease. However, the relation between the Xba I polymorphism and cholesterol gallstones is unknown. This study was aimed at characterizing the polymorphism of the apo B gene Xba I in patients with gallbladder stones and the association of Xba I polymorphism with serum lipids. Xba I genotypes were measured by PCR-RFLP, and serum lipids assayed in 190 patients with gallbladder stones and 441 control subjects. The frequency of the X+/- genotype (20.63 vs. 7.94%) and X+ allele (10.79 vs. 3.97%) was significantly higher in the patient group than in the control group. Patients with the X+/- genotype had a significantly higher concentration of total cholesterol, low-density lipoprotein (LDL)-cholesterol, and apo B in serum than patients with the X-/- genotype. The X+ allele of the apo B gene is characterized by a higher cholesterol concentration and a higher LDL-cholesterol concentration in serum, and it may be a marker for increased risk of cholesterol gallstone disease.

Cholesterol synthesis inhibitors (HMG-CoA Reductase Inhibitors) are reported to decrease cholesterol saturation index of duodenal bile in hypercholesterolaemic subjects. The dissolution of gallstones in animals on treatment with these drugs created expectations of a therapeutical role for these drugs in cholesterol gallstone disease. However, in prospective studies with these drugs in humans, no effect on number and size of cholesterol gallstones was observed. This is likely the result of the fact that not just biliary secretion of cholesterol is decreased during treatment with these drugs in cholesterol gallstone disease, but phospholipids and bile salts as well. As a consequence, nucleation time of cholesterol crystals in gallbladder bile is not influenced by these drugs. Another important determinant in cholesterol gallstone disease, e.g. gallbladder motility, is not influenced by HMG-CoA reductase inhibitors. Although these drugs and their metabolites are secreted into the bile, they do not influence biliary lithogenicity. In conclusion, there seems to be no therapeutic role for HMG-CoA reductase inhibitors in the treatment of cholesterol gallstone disease, although no negative effects on determinants of cholesterol gallstone formation during treatment with these drugs are observed either.