David Q.-H. Wang

miR-33 controls the expression of biliary transporters, and mediates statin- and diet-induced hepatotoxicity

Bile secretion is essential for whole body sterol homeostasis. Loss‐of‐function mutations in specific canalicular transporters in the hepatocyte disrupt bile flow and result in cholestasis. We show that two of these transporters, ABCB11 and ATP8B1, are functional targets of miR‐33, a micro‐RNA that is expressed from within an intron of SREBP‐2. Consequently, manipulation of miR‐33 levels in vivo with adenovirus or with antisense oligonucleotides results in changes in bile secretion and bile recovery from the gallbladder. Using radiolabelled cholesterol, we show that systemic silencing of miR‐33 leads to increased sterols in bile and enhanced reverse cholesterol transport in vivo. Finally, we report that simvastatin causes, in a dose‐dependent manner, profound hepatotoxicity and lethality in mice fed a lithogenic diet. These latter results are reminiscent of the recurrent cholestasis found in some patients prescribed statins. Importantly, pretreatment of mice with anti‐miR‐33 oligonucleotides rescues the hepatotoxic phenotype. Therefore, we conclude that miR‐33 mediates some of the undesired, hepatotoxic effects of statins.

Role of mitochondria in nonalcoholic fatty liver disease-from origin to propagation

OBJECTIVES Mitochondria play a major role in cell energy-generating processes and integrate several signalling pathways to control cellular life and death. DESIGN AND METHODS Several liver diseases are characterized by mitochondrial alterations which are directly or indirectly dependent on the activation of intracellular stress cascades or receptor-mediated pathways. This article examines the role of mitochondrial dysfunction in critical initiating or propagating events in fatty liver infiltration and nonalcoholic fatty liver disease (NAFLD). Genetic variants and the role of drug-induced toxicity have been considered. RESULTS Key alterations of mitochondrial physiology associated with hepatocyte fatty changes are described. The value of novel non-invasive diagnostic methods to detect mitochondrial metabolic alterations is also discussed. CONCLUSIONS Mitochondrial metabolic remodeling is a predominant factor in the appearance and perpetuation of hepatocyte fat accumulation. Non-invasive techniques to identify mitochondrial dysfunction and proper mitochondria protection are two necessary clinical steps for an efficient management of NAFLD.

Gallbladder histopathology during murine gallstone formation: relation to motility and concentrating function.

C57L mice are susceptible and AKR mice are resistant to gallstone formation. We studied in male mice of both strains gallbladder histopathology, cholecystokinin-induced emptying, and concentrating function at 0, 14, 28, and 56 days on a lithogenic diet. Gallbladder wall thickness increased on the diet, with stromal granulocyte infiltration, progressive fibrosis, edema, and epithelial cell indentation, particularly in C57L. Strong basal cholecystokinin octapeptide-induced gallbladder emptying (70% of fasting volumes) occurred in both strains, but fasting gallbladder volumes were significantly larger in C57L (14.8 ± 2.2 μl vs. 8.8 ± 1.0 μl). On the diet, fasting volumes increased exclusively in C57L (28.6 ± 2.9 μl on day 56), with progressively decreased emptying (27% of fasting volumes on day 56). Gallbladder emptying remained normal in AKR. Gallbladder concentrating function decreased on the lithogenic diet (especially in C57L), coinciding with decreased aquaporin-1 (AQP1) and AQP8 expression at the mRNA and protein levels. In additional experiments, similar downregulation of AQP1 and AQP8 mRNA expression occurred in farnesoid X receptor (FXR)-deficient mice after 1 week on the lithogenic diet, without any difference from corresponding wild-type mice. In conclusion, during murine lithogenesis, altered gallbladder histology is associated with impaired motility, reduced concentrating function, and decreased AQP1 and AQP8 expression, the latter without the involvement of the FXR.

Ginsenoside Rb1 reduces fatty liver by activating AMP-activated protein kinase in obese rats

Ginsenoside Rb1 (Rb1), a natural compound extracted from ginseng, exerts anti-obesity activity and improves insulin sensitivity in high-fat diet (HFD)-induced obese rats. The objective of the current study was to evaluate the protective effect of Rb1 on fatty liver in HFD-induced obese rats and to elucidate underlying mechanisms. After chronic intraperitoneal administration, Rb1 (10 mg/kg) significantly ameliorated hepatic fat accumulation in HFD-induced obese rats, as demonstrated by reduced liver weight, hepatic triglyceride content, and histological evaluation of liver sections by hematoxylin and eosin and Oil Red O staining. Using primary cultured rat hepatic cells, we found that the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase 1 (CPT1), a key enzyme in fatty acid β-oxidation, were significantly elevated in Rb1-treated hepatocytes compared with those of vehicle-treated cells. HPLC analysis revealed that Rb1 increased the cellular AMP/ATP ratio, which is associated with elevated activation of hepatic AMP-activated protein kinase (AMPK) and phosphorylated acetyl-CoA carboxylase. Consistent with the activation of AMPK, Rb1 stimulated the expression of genes encoding fatty acid oxidative enzymes and proteins, and suppressed the expression of genes encoding enzymes or proteins that function in lipogenesis, assessed by quantitative PCR. We conclude that Rb1 has a potent ability to reduce hepatic fat accumulation and might be useful as a therapeutic agent for fatty liver disorder.

Molecular pathophysiology and physical chemistry of cholesterol gallstones.

Cholesterol gallstones are one of the most prevalent and most costly digestive diseases in the developed countries. Although precipitation of cholesterol from supersaturated bile is the first irreversible physical-chemical step in cholesterol gallstone formation, hepatic hypersecretion of biliary cholesterol is the primary defect in the formation of cholesterol gallstones. The other common abnormalities of the hepatobiliary system in gallstone patients include accelerated cholesterol nucleation/crystallization, gallbladder hypomotility, hypersecretion and accumulation of mucins, high efficiency of intestinal cholesterol absorption and slow intestinal motility. Family and twin studies in humans as well as gallstone prevalence investigations in different strains of inbred mice have clearly demonstrated that a complex genetic basis could determine individual predisposition to develop cholesterol gallstones in response to environmental factors such as high dietary cholesterol. In this review, we summarize progress in understanding the molecular pathophysiology of cholesterol gallstone formation with particular reference to most recent advances in the physical-chemistry of bile and the physiology of biliary lipid secretion.

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 μm pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Ezetimibe: Its Novel Effects on the Prevention and the Treatment of Cholesterol Gallstones and Nonalcoholic Fatty Liver Disease

The cholesterol absorption inhibitor ezetimibe can significantly reduce plasma cholesterol concentrations by inhibiting the Niemann-Pick C1-like 1 protein (NPC1L1), an intestinal sterol influx transporter that can actively facilitate the uptake of cholesterol for intestinal absorption. Unexpectedly, ezetimibe treatment also induces a complete resistance to cholesterol gallstone formation and nonalcoholic fatty liver disease (NAFLD) in addition to preventing hypercholesterolemia in mice on a Western diet. Because chylomicrons are the vehicles with which the enterocytes transport cholesterol and fatty acids into the body, ezetimibe could prevent these two most prevalent hepatobiliary diseases possibly through the regulation of chylomicron-derived cholesterol and fatty acid metabolism in the liver. It is highly likely that there is an intestinal and hepatic cross-talk through the chylomicron pathway. Therefore, understanding the molecular mechanisms whereby cholesterol and fatty acids are absorbed from the intestine could offer an efficacious novel approach to the prevention and the treatment of cholesterol gallstones and NAFLD.

Mitochondria in chronic liver disease.

Mitochondria are the main energy source in hepatocytes and play a major role in extensive oxidative metabolism and normal function of the liver. This key role also assigns mitochondria a gateway function in the center of signaling pathways that mediate hepatocyte injury, because impaired mitochondrial functions affect cell survival and contribute to the onset and perpetuation of liver diseases. Altered mitochondrial functions have indeed been documented in a variety of chronic liver diseases including alcohol-induced liver disease, nonalcoholic fatty liver disease, viral hepatitis, primary and secondary cholestasis, hemochromatosis, and Wilsons disease. Major changes include impairment of the electron transport chain and/or oxidative phosphorylation leading to decreased oxidative metabolism of various substrates, decreased ATP synthesis, and reduced hepatocyte tolerance towards stressing insults. Functional impairment of mitochondria is often accompanied by structural changes, resulting in organelle swelling and formation of inclusion in the mitochondrial matrix. Adequate mitochondrial functions in hepatocytes are maintained by mitochondrial proliferation and/or increased activity of critical enzymes. The assessment of mitochondrial functions in vivo can be a useful tool in liver diseases for diagnostic and prognostic purposes, and also for the evaluation of (novel) therapeutic interventions.

Therapy of gallstone disease: What it was, what it is, what it will be

Cholesterol gallstone disease is a common clinical condition influenced by genetic factors, increasing age, female gender, and metabolic factors. Although laparoscopic cholecystectomy is currently considered the gold standard in treating patients with symptomatic gallstones, new perspectives regarding medical therapy of cholelithiasis are currently under discussion, also taking into account the pathogenesis of gallstones, the natural history of the disease and the analysis of the overall costs of therapy. A careful selection of patients may lead to successful non-surgical therapy in symptomatic subjects with a functioning gallbladder harboring small radiolucent stones. The classical oral litholysis by ursodeoxycholic acid has been recently paralleled by new experimental observations, suggesting that cholesterol-lowering agents which inhibit cholesterol synthesis (statins) or intestinal cholesterol absorption (ezetimibe), or drugs acting on specific nuclear receptors involved in cholesterol and bile acid homeostasis, might be proposed as additional approaches for treating cholesterol gallstones. In this review we discuss old, recent and future perspectives on medical treatment of cholesterol cholelithiasis.

Obesity and the risk and prognosis of gallstone disease and pancreatitis

Obesity is a risk factor for the formation of cholesterol gallstones and exposes patients to increased risk of gallstone-related complications and cholecystectomy. Rapid weight loss achieved by very low calorie diets or bariatric surgery is also a risk factor for cholelithiasis in obese patients, and therapy should take into account the higher prevalence of gallstones, the possibility of more frequent complications and the need for prophylactic treatment with oral ursodeoxycholic acid during weight loss. Obesity is also frequent in children and adolescents, and the burden of cholesterol cholelithiasis is increasing in this population. The chance to develop acute pancreatitis and the severity of the disease are higher in obese subjects because of specific pathogenic factors, including supersaturated bile and crystal formation, rapid weight loss, and visceral obesity. All health policies aimed at reducing the incidence of obesity worldwide will decrease the incidence of gallstones and gallstone-related complications. The pathophysiological scenarios and the therapeutic implications for obesity, gallstone disease, and pancreatitis are discussed.