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Unlabelled: Because hyperhomocysteinemia can occur in cholesterol gallstone disease, we hypothesized that this may result from trimethylation of phosphatidylethanolamine (PE), which partakes in biliary phosphatidylcholine (PC) hypersecretion during cholesterol cholelithogenesis. We fed murine strains C57L/J, C57BL/6J, SWR/J, AKR/J, PE N-methyltransferase (PEMT) knockout (KO), PEMT heterozygous (HET), and wildtype (WT) mice a cholesterol/cholic acid lithogenic diet (LD) for up to 56 days and documented biliary lipid phase transitions and secretion rates. We quantified plasma total homocysteine (tHcy), folate, and vitamin B12 in plasma and liver, as well as biliary tHcy and cysteine secretion rates. Rate-limiting enzyme activities of PC synthesis, PEMT and cytidine triphosphate: phosphocholine cytidylyltransferase (PCT), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in liver homogenates. Other potential sources of plasma tHcy, glycine N-methyltransferase (GNMT) and guanidinoacetate N-methyltransferase (GAMT), were assayed by gene expression. Plasma tHcy and PEMT activities became elevated during cholelithogenesis in gallstone-susceptible C57L, C57BL/6, and SWR mice but not in the gallstone-resistant AKR mice. Persisting in C57L mice, which exhibit the greatest Lith gene burden, these increases were accompanied by elevated hepatic SAM/SAH ratios and augmented biliary tHcy secretion rates. Counter-regulation included remethylation of Hcy to methionine concurrent with decreased folate and vitamin B12 levels and Hcy transsulfuration to cysteine. Concomitantly, methylenetetrahydrofolate reductase (Mthfr), betaine-homocysteine methyltransferase (Bhmt), and cystathionine-β-synthase (Cbs) were up-regulated, but Gnmt and Gamt genes were down-regulated. PEMT KO and HET mice displayed biliary lipid secretion rates and high gallstone prevalence rates similar to WT mice without any elevation in plasma tHcy levels., Conclusion: This work implicates up-regulation of PC synthesis by the PEMT pathway as a source of elevated plasma and bile tHcy during cholesterol cholelithogenesis., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Long-Evans Cinnamon (LEC) rats exhibit a genetic defect in Atp7b gene, which is homologous to the human Wilson's disease gene, resulting in an inability to mobilize copper from the liver. This study was undertaken to gain insight into the relationship between liver copper accumulation and plasma lipid profile, circulating lipoprotein composition, hepatic sterol metabolism and biliary lipid secretion rates in 12-week-old LEC rats compared to control Long-Evans rats. Concomitant with hepatic copper deposition, LEC rats displayed increased content of triglycerides (TGs), free cholesterol (FC) and cholesteryl ester (CE) in the liver. Hepatic concentrations of malondialdehyde (MDA), an index of lipid peroxidation were also significantly elevated in LEC rats (50%). This steatosis was associated with aberrant microsomal apolipoprotein (apo) B-100 and microsomal triglyceride transfer protein (MTP) content, hypotriglyceridemia, hypocholesterolemia and abnormalities in both circulating lipoprotein composition and size. Atypical hepatobiliary sterol metabolism was established by the assessment of the activity of key intracellular enzymes for cholesterol homeostasis, which demonstrated, with respect to controls, a 40% reduction in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a 30% reduction in cholesterol 7alpha-hydroxylase, and a 54% reduction in acyl CoA:cholesterol acyltransferase. During a 6-h biliary drainage, a decline in the bile acid output was recorded and might be linked to the low protein expression of the bile salt export pump (BSEP or ABCB11). Our data emphasize the crucial role of copper balance in hepatic sterol homeostasis and lipoprotein metabolism in LEC rats. Additional studies are needed to delineate the mechanisms of these disorders.

Background & Aims: Helicobacter spp are common inhabitants of the hepatobiliary and gastrointestinal tracts of humans and animals and cause a variety of well-described diseases. Recent epidemiologic results suggest a possible association between enterohepatic Helicobacter spp and cholesterol cholelithiasis, chronic cholecystitis, and gallbladder cancer. To test this, we prospectively investigated the effects of Helicobacter spp infection in cholesterol gallstone pathogenesis in the highly susceptible C57L/J mouse model., Methods: Helicobacter spp-free adult male C57L mice were infected with several different enterohepatic Helicobacter spp or left uninfected and fed either a lithogenic diet or standard mouse chow for 8 and 18 weeks. At the conclusion of the study, bile was examined microscopically and diagnostic culture and polymerase chain reaction were performed., Results: Mice infected with Helicobacter bilis or coinfected with Helicobacter hepaticus and Helicobacter rodentium and fed a lithogenic diet developed cholesterol gallstones at 80% prevalence by 8 weeks compared with approximately 10% in uninfected controls. Monoinfections with H hepaticus , Helicobacter cinaedi , and H rodentium gave a cholesterol gallstone prevalence of 40%, 30%, and 20%, respectively; the latter 2 groups did not differ significantly from uninfected animals. Neither infected nor uninfected mice fed a chow diet developed cholesterol gallstones., Conclusions: These findings, along with prior epidemiologic studies, suggest that Helicobacter spp play a major role in the pathophysiology of cholesterol gallstone formation in mice and perhaps humans.

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.

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.

Glutathione appears to be a major osmotic factor in the generation of bile salt-independent flow (BSIF). This study was designed to investigate its importance in the pathology of 17-alpha-ethinyl estradiol (EE)-induced cholestasis. Five-day EE treatment at the dose level of 5 mg/kg/day significantly decreased bile flow (57% of controls) and biliary glutathione secretion. Evaluation of the contribution of bile salt dependent flow (BSDF), glutathione dependent flow (GSDF) and the bile flow generated independently of both bile salts and glutathione (BS-GSIF) revealed that EE decreased all portions of the flow (63, 44 and 52% of control values, respectively). At 4 and 20 h after a single administration of the same EE dose, a significant diminution of bile flow was noted (decreases of 17 and 29%, respectively) in association with a significant fall in biliary glutathione content. Under these conditions, BSDF and BS-GSIF were not modified (98 and 112% of control BSDF values, respectively; 96 and 99% of control BS-GSIF values, respectively) while GSDF was decreased markedly, representing 65 and 50% of control values. Biliary glutathione secretion was diminished without modification of liver and blood glutathione concentration or redox status following single EE dose whereas, after 5 days of EE treatment, a significant increase in liver glutathione was observed, suggesting that EE may interfere with the glutathione secretory process. This study demonstrates that EE rapidly alters biliary glutathione content, leading to a marked decline in GSDF. This reduction may explain the decrease in BSIF produced by EE at the outset of cholestasis.

This study examined the effect of moderate long-term exercise, begun soon after weaning, on the age-related decline in bile formation and the secretory rate maximum (SRm) of taurocholate (TC), the major bile salt (BS) in rats. Eight-month-old sedentary (S) female Sprague-Dawley rats showed a significant decrease in basal and TC-stimulated bile formation when compared with 2.5-month-old S controls. As in younger rats, decreased biliary phospholipid (PL) output was associated with the TC SRm, but this change appeared much more rapidly in S animals, which also exhibited significant increased plasma lipids and higher TC concentrations in plasma and liver at the end of the TC infusion. Exercise significantly improved bile flow (BF), including the bile salt-dependent and -independent fractions under basal and TC-stimulated conditions in 8 month-old rats. Although all the biliary parameters evaluated were improved, maximal BF and PL secretion values were affected the most and were virtually not different from those obtained in younger S animals. Exercise also significantly lowered the age-related elevation of plasma PL. Thus, moderate long-term exercise exerts a beneficial effect on hepatobiliary function and the BS SRm, an effect that may be attributed in part to increased availability of a biliary PL pool previously implicated in regulation of the BS SRm.

We investigated the contribution of bile salts and glutathione (GSH) to the generation of bile flow in young, mature, and old female Sprague-Dawley rats, either fed ad libitum (AL) or subjected to a 40% dietary restriction (DR), which was supplemented or not with vitamins and minerals, starting from weaning. An age-related decline in bile flow was observed in the AL group. DR increased bile flow compared to age-matched AL rats, resulting in a twofold increase in the old animals. This was associated with a statistically significantly higher biliary GSH secretion rate and a moderate increase in the bile salt secretory rate. The apparent GSH-dependent flow was significantly increased in DR groups of all ages. Hepatic GSH concentration was closely related to the GSH secretion rate. These results indicate that the increase in biliary GSH content produced by DR is the major mediator of the increased bile flow, resulting in enhanced GSH and GSH-derived thiols supply to the intestinal lumen. [ABSTRACT FROM AUTHOR]