Giuseppe Palasciano

Cholesterol gallstone disease.

With a prevalence of 10-15% in adults in Europe and the USA, gallstones are the most common digestive disease needing admission to hospital in the West. The interplay between interprandial and postprandial physiological responses to endogenous and dietary lipids underscores the importance of coordinated hepatobiliary and gastrointestinal functions to prevent crystallisation and precipitation of excess biliary cholesterol. Indeed, identifying the metabolic and transcriptional pathways that drive the regulation of biliary lipid secretion has been a major achievement in the field. We highlight scientific advances in protein and gene regulation of cholesterol absorption, synthesis, and catabolism, and biliary lipid secretion with respect to the pathogenesis of cholesterol gallstone disease. We discuss the physical-chemical mechanisms of gallstone formation in bile and the active role of the gallbladder and the intestine. We also discuss gaps in our knowledge of the pathogenesis of gallstone formation and the potential for gene targeting in therapy.

Selective Activation of Nuclear Bile Acid Receptor FXR in the Intestine Protects Mice Against Cholestasis

BACKGROUND & AIMS Cholestasis is a liver disorder characterized by impaired bile flow, reduction of bile acids (BAs) in the intestine, and retention of BAs in the liver. The farnesoid X receptor (FXR) is the transcriptional regulator of BA homeostasis. Activation of FXR by BAs reduces circulating BA levels in a feedback mechanism, repressing hepatic cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme for the conversion of cholesterol to BAs. This mechanism involves the hepatic nuclear receptor small heterodimer partner and the intestinal fibroblast growth factor (FGF) 19 and 15. We investigated the role of activation of intestine-specific FXR in reducing hepatic levels of BAs and protecting the liver from cholestasis in mice. METHODS We generated transgenic mice that express a constitutively active FXR in the intestine. Using FXR gain- and loss-of-function models, we studied the roles of intestinal FXR in mice with intrahepatic and extrahepatic cholestasis. RESULTS Selective activation of intestinal FXR induced FGF15 and repressed hepatic Cyp7a1, reducing the pool size of BAs and changing the BA pool composition. Activation of intestinal FXR protected mice from obstructive extrahepatic cholestasis after bile duct ligation or administration of α-naphthylisothiocyanate. In Mdr2(-/-) mice, transgenic expression of activated FXR in the intestine protected against liver damage, whereas absence of FXR promoted progression of liver disease. CONCLUSIONS Activation of FXR transcription in the intestine protects the liver from cholestasis in mice by inducing FGF15 expression and reducing the hepatic pool of BA; this approach might be developed to reverse cholestasis in patients.

Silybin combined with phosphatidylcholine and vitamin E in patients with nonalcoholic fatty liver disease: A randomized controlled trial

The only currently recommended treatment for nonalcoholic fatty liver disease (NAFLD) is lifestyle modification. Preliminary studies of silybin showed beneficial effects on liver function. Realsil (RA) comprises the silybin phytosome complex (silybin plus phosphatidylcholine) coformulated with vitamin E. We report on a multicenter, phase III, double-blind clinical trial to assess RA in patients with histologically documented NAFLD. Patients were randomized 1:1 to RA or placebo (P) orally twice daily for 12 months. Prespecified primary outcomes were improvement over time in clinical condition, normalization of liver enzyme plasma levels, and improvement of ultrasonographic liver steatosis, homeostatic model assessment (HOMA), and quality of life. Secondary outcomes were improvement in liver histologic score and/or decrease in NAFLD score without worsening of fibrosis and plasma changes in cytokines, ferritin, and liver fibrosis markers. We treated 179 patients with NAFLD; 36 were also HCV positive. Forty-one patients were prematurely withdrawn and 138 patients analyzed per protocol (69 per group). Baseline patient characteristics were generally well balanced between groups, except for steatosis, portal infiltration, and fibrosis. Adverse events (AEs) were generally transient and included diarrhea, dysgeusia, and pruritus; no serious AEs were recorded. Patients receiving RA but not P showed significant improvements in liver enzyme plasma levels, HOMA, and liver histology. Body mass index normalized in 15% of RA patients (2.1% with P). HCV-positive patients in the RA but not the P group showed improvements in fibrogenesis markers. This is the first study to systematically assess silybin in NAFLD patients. Treatment with RA but not P for 12 months was associated with improvement in liver enzymes, insulin resistance, and liver histology, without increases in body weight. These findings warrant further investigation.

Ribavirin-induced anemia: mechanisms, risk factors and related targets for future research.

Ribavirin (RBV) is an antiviral nucleoside analogue commonly used in combination with interferon for the treatment of chronic hepatitis C. Severe anemia develops in about 10% of treated patients, and requires close monitoring of hemoglobin and often RBV dose reduction, which may compromise sustained virologic response. Anemia is likely related to extensive RBV accumulation in erythrocytes subsequent to active unidirectional transmembraneous transport. RBV exerts its toxicity through an inhibition of intracellular energy metabolism and oxidative membrane damage, leading to an accelerated extravascular hemolysis by the reticulo-endothelial system. Concentration-dependent toxicity and improvement of anemia upon dose-reduction point towards the importance of pharmacokinetic factors for RBV-induced anemia. On the other hand, pronounced variability in the correlation between RBV concentration and Hb reduction limits the prediction of anemia based on plasma or erythrocyte concentrations in individual patients and points towards additional factors determining individual susceptibility to RBV-induced anemia. Recent studies suggest that erythrocyte oxidative defense mechanisms may play an important role in RBV-induced anemia. Clinical risk factors for severe RBV-induced anemia include impaired renal function, high age, high dose per body weight and female gender. Determination of RBV concentrations has little value in the management of anemia. The only proven effective prevention of RBV-induced anemia is the concomitant administration of erythropoietin. Future research on RBV pharmacokinetics and pharmacodynamics, as well as erythrocyte antioxidant defense mechanisms may improve safety and efficacy of RBV therapy and guide the development of new treatments for RBV-induced anemia and alternative antiviral agents.

Acute ethanol administration induces oxidative changes in rat pancreatic tissue.

BACKGROUND--There is mounting clinical evidence that ethanol toxicity to the pancreas is linked with glutathione depletion from oxidative stress but there is not experimental proof that this occurs. AIMS AND METHODS--The effect of acute ethanol ingestion (4 g/kg) on the pancreatic content of reduced (GSH) and oxidised (GSSG) glutathione, malondialdehyde (MDA), and carbonyl proteins were therefore studied in the rat. RESULTS--Ethanol caused a significant reduction in GSH (p < 0.02) and an increase in GSSG (p < 0.005), MDA (p < 0.05), and carbonyl proteins (p < 0.05) in the rat pancreas. The GSH/GSSG ratios were significantly decreased after ethanol, especially in rats pretreated with diethylmaleate (DEM), a GSH blocker. Administration of ethanol after DEM further increased the rate of lipid and protein oxidation. Pretreatment with cyanamide (an inhibitor of aldehyde dehydrogenase) but not with 4-methylpyrazole (an alcohol dehydrogenase inhibitor) caused higher production of GSSG and MDA. CONCLUSIONS--These findings indicate that acute ethanol reduces the pancreatic content of GSH, which seems to be protective against ethanol toxicity, since its depletion is accompanied by increased oxidative damage to cell structures. The further increase of lipid peroxidation and GSSG production in the presence of cyanamide suggests that acetaldehyde might be responsible for the oxidative changes that occur in pancreatic cells after ethanol administration.

Bile acids and colon cancer: Solving the puzzle with nuclear receptors

Colorectal cancer is the third most common malignancy worldwide and is often linked to obesity, a sedentary lifestyle, carbohydrate- and fat-rich diets and elevated fecal excretion of secondary bile acids. Accumulation of toxic bile acids triggers oxidative damage, mitochondrial dysfunction and tumor progression. Nuclear receptors are transcription factors crucially involved in the regulation of bile acid metabolism and detoxification, and their activation may confer protection from bile acid tumor-promoting activity. In this review, we explore the tangled relationships among bile acids, nuclear receptors and the intestinal epithelium, with particular emphasis on the role of the farnesoid X receptor in colorectal cancer prevention and on novel nuclear receptor-based approaches to expand the portfolio of chemotherapeutic agents.

Cell type-dependent effect of phospholipid and cholesterol on bile salt cytotoxicity

The effect of phosphatidylcholine and cholesterol on bile salt-induced cytotoxicity was investigated. Experiments were performed in both human erythrocytes and cultured CaCo-2 cells, a model system for gastrointestinal epithelium. Hemolysis induced by 50 mmol/L sodium-taurocholate was reduced by both lecithin and cholesterol in a concentration-dependent manner. Cholesterol was 10 times more effective than phosphatidylcholine. Addition of only small amounts of the sterol to phosphatidylcholine/taurocholate solutions eliminated all cytotoxicity. The protective influence of cholesterol is probably mediated through a direct effect on the membrane. Incubation of erythrocytes with a cholesterol/taurocholate mixture greatly increased the cholesterol content of the membrane. With respect to sensitivity to bile salts and the protective effect of lecithin, CaCo-2 cells behaved very similar to erythrocytes. However, cholesterol failed to have any cytoprotective effect when used in combination with taurocholate or phosphatidylcholine/taurocholate solutions. Interestingly, at relatively high concentrations of cholesterol (cholesterol saturation index greater than 1.0), the sterol even increased cytotoxicity. This correlated with a cholesterol-induced shift of phosphatidylcholine from micelles to vesicles, which normally occurs in supersaturated model and human bile. The different sensitivity of the two cell types to the effect of cholesterol on bile salt damage might be caused by the difference in lipid membrane composition. In conclusion, CaCo-2 cells represent a physiologically more relevant model system to study bile cytotoxicity than erythrocytes. When extrapolated to gallbladder epithelial cells, these results could be relevant for the pathogenesis of gallstone disease. The increased cytotoxicity might be the signal by means of which supersaturated bile induces mucin hypersecretion by gallbladder epithelial cells.

Coordinate regulation of gallbladder motor function in the gut‐liver axis

Gallstones are one of the most common digestive diseases with an estimated prevalence of 10%‐15% in adults living in the western world, where cholesterol‐enriched gallstones represent 75%‐80% of all gallstones. In cholesterol gallstone disease, the gallbladder becomes the target organ of a complex metabolic disease. Indeed, a fine coordinated hepatobiliary and gastrointestinal function, including gallbladder motility in the fasting and postprandial state, is of crucial importance to prevent crystallization and precipitation of excess cholesterol in gallbladder bile. Also, gallbladder itself plays a physiopathological role in biliary lipid absorption. Here, we present a comprehensive view on the regulation of gallbladder motor function by focusing on recent discoveries in animal and human studies, and we discuss the role of the gallbladder in the pathogenesis of gallstone formation. (HEPATOLOGY 2008;47:2112–2126.)

Gallbladder motor function in gallstone patients: sonographic and in vitro studies on the role of gallstones, smooth muscle function and gallbladder wall inflammation

Gallbladder motility was studied by ultrasound in 100 healthy adult volunteers and 150 gallstone patients, in a subgroup of whom gallstone burden, type and number, gallbladder histology and tensiometric responses of gallbladder strips to cholecystokinin octapeptide were evaluated. Patients were divided into contractors (n = 108) and hypocontractors (n = 42), according to their gallbladder motility pattern in vivo. Contractors showed slower gallbladder emptying and increased fasting and postprandial residual volumes, although the ejected amount of bile was greater than that of controls (20.2 +/- SEM 1.1 vs 16.0 +/- 0.7 ml; p < 0.001). In contrast, hypocontractors exhibited slower and less complete gallbladder emptying than controls with a reduction in the absolute amount of ejected bile. Although gallbladder wall inflammation was mild and comparable in specimens from both groups of patients, the thickness of the muscular layer was greater in hypocontractors than in contractors (1073 +/- 76 vs 745 +/- 75 microns, p < 0.01) and related inversely to postprandial ejected volume (r = -0.42; p < 0.03; n = 32) but positively to gallstone volume (r = 0.40; p < 0.03; n = 32). Compared to contractors, gall-bladder muscle strips of hypocontractors exhibited a decrease in frequency and amplitude of spontaneous contraction and in maximal stress and receptor sensitivity to cholecystokinin octapeptide (0.1 nM-1 microM). Postprandial gallbladder evaculation was unaffected by stone number, and by the presence or absence of stone calcification. Gallstone volume was larger in hypocontractors (19.4 +/- 3.0 ml vs 9.6 +/- 0.9 ml, p < 0.001) than contractors. The comparison of in vitro contractility patterns between cholesterol, mixed and pigment stone patients showed a more severe defect in patients with cholesterol and mixed stones than in those with pigment calculi. In conclusion, in gallstone patients: (i) gallbladder motor dysfunction manifests mainly with increased fasting and postprandial residual volumes in contractors and with markedly increased postprandial residual volumes and decreased gallbladder emptying in hypocontractors; (ii) gallbladder kinetics seem to be influenced by stone volume and cholesterol content of calculi but not stone number, calcification or mild chronic cholecystitis; (iii) a form of hypertrophic leiomyopathy is observed in gallstone patients with the most impaired gallbladder motor function.

Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis

Effectively assessing subtle hepatic metabolic functions by novel non-invasive tests might be of clinical utility in scoring NAFLD (non-alcoholic fatty liver disease) and in identifying altered metabolic pathways. The present study was conducted on 39 (20 lean and 19 obese) hypertransaminasemic patients with histologically proven NAFLD {ranging from simple steatosis to severe steatohepatitis [NASH (non-alcoholic steatohepatitis)] and fibrosis} and 28 (20 lean and eight overweight) healthy controls, who underwent stable isotope breath testing ([(13)C]methacetin and [(13)C]ketoisocaproate) for microsomal and mitochondrial liver function in relation to histology, serum hyaluronate, as a marker of liver fibrosis, and body size. Compared with healthy subjects and patients with simple steatosis, NASH patients had enhanced methacetin demethylation (P=0.001), but decreased (P=0.001) and delayed (P=0.006) ketoisocaproate decarboxylation, which was inversely related (P=0.001) to the degree of histological fibrosis (r=-0.701), serum hyaluronate (r=-0.644) and body size (r=-0.485). Ketoisocaproate decarboxylation was impaired further in obese patients with NASH, but not in patients with simple steatosis and in overweight controls. NASH and insulin resistance were independently associated with an abnormal ketoisocaproate breath test (P=0.001). The cut-off value of 9.6% cumulative expired (13)CO(2) for ketoisocaproate at 60 min was associated with the highest prediction (positive predictive value, 0.90; negative predictive value, 0.73) for NASH, yielding an overall sensitivity of 68% and specificity of 94%. In conclusion, both microsomal and mitochondrial functions are disturbed in NASH. Therefore stable isotope breath tests may usefully contribute to a better and non-invasive characterization of patients with NAFLD.