Ludwig Amigo

Ezetimibe prevents cholesterol gallstone formation in mice.

Background: Intestinal cholesterol absorption may influence gallstone formation and its modulation could be a useful therapeutic strategy for gallstone disease (GSD). Ezetimibe (EZET) is a cholesterol‐lowering agent that specifically inhibits intestinal cholesterol absorption.

Impaired biliary cholesterol secretion and decreased gallstone formation in apolipoprotein E–Deficient mice fed a high-cholesterol diet

BACKGROUND & AIMS Because apolipoprotein E (apoE) is a key cholesterol transport molecule involved in the hepatic uptake of chylomicron cholesterol, it may play a critical role in controlling bile cholesterol elimination and cholesterol gallstone formation induced by dietary cholesterol. To test this hypothesis, we studied biliary lipid secretion and gallstone formation in apoE-deficient mice fed cholesterol-rich diets. METHODS Bile lipid outputs and gallstone sequence events were analyzed in apoE-deficient mice fed a high-cholesterol diet or a lithogenic diet compared with control animals. RESULTS A high-cholesterol diet increased biliary cholesterol secretion and gallbladder bile cholesterol concentration in wild-type mice; the increase in bile cholesterol secretion was significantly attenuated in apoE-deficient mice. ApoE knockout mice fed a high-cholesterol lithogenic diet had a markedly lower frequency of gallbladder bile cholesterol crystal and gallstone formation than wild-type mice, which was most likely a result of the decreased cholesterol saturation index found in gallbladder bile of apoE-deficient mice. CONCLUSIONS These results show that apoE expression is an important factor for regulating both biliary secretion of diet-derived cholesterol as well as diet-induced cholesterol gallstone formation in mice.

Protective role of biliary cholesterol and phospholipid lamellae against bile acid-induced cell damage

BACKGROUND/AIMS Bile salts (BS) are cytotoxic agents, but cell damage is not observed in the hepatobiliary system. We hypothesized that biliary lipid vesicles (unilamellae and multilamellae) could have a protective role against BS-induced cytotoxicity. METHODS Biliary lipid lamellar secretion was induced by feeding rats with 0.5% diosgenin. Cytoprotection was assessed in bile duct-obstructed rats and by incubating human erythrocytes with sodium taurocholate. RESULTS Biliary cholesterol concentration increased > 300% in diosgenin-fed rats; electron microscopic examination showed a great abundance of lipid lamellar vesicles in bile and within the canaliculi. After bile duct obstruction, serum hepatic enzyme activities were significantly lower in diosgenin-fed rats. Histologically severe and confluent hepatocellular necrosis was only observed in control rats. Biliary lamellar lipid material significantly reduced the BS-induced hemolytic effect in vitro in a concentration-dependent manner. This protective effect correlated to a progressive decrease in the intermicellar BS concentration. Phosphatidylcholine or cholesterol, alone or as lamellar structures, also showed cytoprotective effect in vitro but always less than native biliary lamellae. CONCLUSIONS These results support the concept that native biliary cholesterol phospholipid lamellae represent an important cytoprotective factor for hepatocytes and biliary epithelial cells against BS-induced damage.

Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile

BACKGROUND & AIMS Biliary proteins are promoters of cholesterol crystallization in artificial model bile. However, their pathogenic importance for cholesterol precipitation in native gallbladder bile (GB) is uncertain. The aim of this study was to evaluate the significance of biliary lipids and proteins on cholesterol crystal detection time (ChCDT) of GB in patients with gallstones. METHODS ChCDT and concentrations of lipids, albumin, mucins, aminopeptidase N, alpha1-acid glycoprotein, haptoglobin, and immunoglobulins (Igs) were measured in GB of 92 patients, 52 of whom had cholesterol gallstones. RESULTS ChCDT was markedly reduced in gallstone patients. Compared with patients without gallstones, they had a significant increase in cholesterol saturation and total protein, albumin, mucin, and IgG biliary concentrations. In univariate analysis, ChCDT of GB was significantly correlated with cholesterol saturation and total lipid, protein, Ig, aminopeptidase N, and alpha1-acid glycoprotein concentrations. However, stepwise logistic regression analysis showed that only cholesterol saturation independently correlated to ChCDT. Gallbladder inflammation correlated with the concentration of Igs, but subtraction of IgG from GB did not modify the ChCDT. CONCLUSIONS Biliary cholesterol transport and saturation, but not proteins, appear critical for the cholesterol crystallization abnormality observed in native bile from patients with gallstones.

Modulation of intrahepatic cholesterol trafficking: evidence by in vivo antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transport into rat bile

Biliary cholesterol represents one of the two major excretory pathways for sterol elimination from the body and plays a central role in cholesterol gallstone formation. Biliary cholesterol originates from a precursor pool of preformed and newly synthesized free cholesterol. Although it has been suggested that newly synthesized and preformed biliary cholesterol are secreted by independent pathways, the specific cellular and molecular mechanisms are unknown. We used male Wistar rats to study the time-course of the appearance of newly synthesized cholesterol, phosphatidylcholine and protein into bile. The specific role of sterol carrier protein-2 (SCP-2) in the transport of newly synthesized biliary cholesterol was evaluated by an in vivo antisense oligonucleotide approach. In contrast to [14C]phosphatidylcholine and [35S]proteins, the time-course of [14C]cholesterol appearance into bile was rapid, and microtubule- and Golgi-independent. In vivo SCP-2 antisense treatment reduced and delayed the appearance of biliary [14C]cholesterol. Furthermore, hepatic SCP-2 expression increased more than 3-fold over control values in rats that had been treated with diosgenin to increase biliary secretion of newly synthesized cholesterol. These results suggest that SCP-2 is necessary for the rapid transport of newly synthesized cholesterol into bile and that hepatocytes can induce SCP-2 expression according to the rate of biliary secretion of newly synthesized cholesterol.

NPC2 is expressed in human and murine liver and secreted into bile: Potential implications for body cholesterol homeostasis†

The liver plays a critical role in the metabolism of lipoprotein cholesterol and in controlling its elimination through the bile. Niemann‐Pick type C 2 (NPC2), a cholesterol‐binding protein, is key for normal intracellular trafficking of lipoprotein cholesterol, allowing its exit from the endolysosomal pathway into the metabolically active pool of the cell. In addition, NPC2 is a secretory protein from astrocytes and epididymal cells. Although NPC2 mRNA is detected in the liver, plasma and biliary NPC2 protein levels and function have not been reported. This study demonstrates that NPC2 is present in murine and human plasma and bile. In addition, hepatic NPC2 protein expression was dramatically increased in NPC1‐deficient mice but not regulated by cholesterol feeding or pharmacological modulation of various nuclear receptors involved in cholesterol and bile acid metabolism. Interestingly, biliary NPC2 levels were 3‐fold increased in gallstone‐susceptible C57BL6/J versus gallstone‐resistant BALB/c mice. Furthermore, NPC2 was exclusively found in the cholesterol pro‐nucleating ConA‐binding fraction of human bile. In conclusion, NPC2 is secreted from the liver into bile and plasma, where it may have a functional role in cholesterol transport in normal and disease conditions. (HEPATOLOGY 2006;43:126–133.)

Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids.

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle‐associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase‐N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase‐N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 μg/ml. Aminopeptidase‐N showed a concentration‐dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase‐N may play a critical role in the pathogenesis of cholesterol gallstone disease.

Cholecystectomy increases hepatic triglyceride content and very-low-density lipoproteins production in mice

Background & aims: Bile acid (BA) pool size remains unchanged after cholecystectomy (XGB) but it circulates faster, exposing the enterohepatic system to an increased flux of BA. Triglyceride (TG) and BA metabolisms are functionally inter‐related. We investigated whether ablation of the gallbladder (GB) modifies hepatic TG metabolism.

Increased activity of hepatic microsomal triglyceride transfer protein and bile acid synthesis in gallstone disease

A strong interrelationship exists between the regulation of bile acid (BA) metabolism and hepatic very low density lipoprotein (VLDL) production. We have recently shown that BA synthesis is increased in gallstone disease. We investigated the activity of hepatic microsomal triglyceride transfer protein (MTTP) as a surrogate of VLDL production, BA synthesis, and mRNA expression levels of proteins that regulate fatty acid (FA) metabolism in the liver of gallstone (GS) patients compared with GS‐free patients. Twenty‐seven volunteers subjected to elective surgery; 9 were GS‐free and 18 with GS agreed to have a liver biopsy. We quantified by a fluorescence assay the activity of MTTP and by quantitative reverse‐transcription PCR (RT‐PCR) the mRNA content of hepatic MTTP and genes that regulate hepatic sterol and FA metabolism. Plasma was assayed for lathosterol and 7α‐hydroxy‐4‐cholesten‐3‐one. Liver histology was normal in GS and GS‐free patients. Serum VLDL triglycerides and apoB were significantly increased in GS. Hepatic triglycerides tripled in GS (P < 0.001) compared with GS‐free. MTTP activity increased 70% (P < 0.001). Serum lathosterol and hepatic cholesterol concentrations, and mRNA expressions of MTTP, CD36, and FABP1 were similar in GS‐free and GS patients. Hepatic mRNA expression of hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGR) and 3‐hydroxyl‐3‐methyl‐glutaryl‐CoA synthase (HMGS) were significantly decreased—40% and 27%, respectively—in GS. Serum 7α‐hydroxy‐4‐cholesten‐3‐one was 75% higher, and mRNA expression of CYP7A1 was increased sevenfold (P < 0.001) in GS. Conclusion: Hepatic MTTP activity and BA synthesis are increased in GS. Results suggest that hepatic VLDL production and trafficking of BA are increased in gallstone patients. (HEPATOLOGY 2007;45:1261–1266.)

Expression and regulation of scavenger receptor class B type I (SR-BI) in gall bladder epithelium

Background and aims: Biliary lipid absorption by the gall bladder mucosa and the cholesterol content of the gall bladder wall appear to play a role in cholesterol gall stone formation. As the scavenger receptor class B type I (SR- BI) regulates cellular cholesterol uptake, we studied its expression in human and murine gall bladders, its regulation by increased biliary lipid content, and its role in gall stone formation. Methods and results: Using immunohistochemistry, SR-BI was found in the apical domain of human gall bladder epithelial cells. Immunoblotting of isolated membranes from gall bladder epithelial cells showed a specific signal for the 82 kDa SR-BI protein. In C57BL/6 mice, SR-BI was also found in the gall bladder epithelium. Using western blot analysis, an inverse relationship was observed between biliary cholesterol concentration and SR-BI expression in murine gall bladder mucosa. By comparing lithogenic diet fed wild-type and SR-BI deficient mice, gall bladder wall cholesterol content and gall stone formation were not found to be dependent on SR-BI expression. Conclusions: (i) SR-BI is expressed in both human and murine gall bladder epithelium; (ii) biliary cholesterol hypersecretion is associated with decreased gall bladder SR-BI expression in mice; and (iii) murine SR-BI is not essential in controlling gall bladder wall cholesterol content and gall stone formation during diet induced cholelithiasis.