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Previous experiments in mice showed that dietary trans-fats could play a role in non-alcoholic steatohepatitis (NASH) yet little is known about the accumulation trans-fats in hepatic lipid pools in relationship to liver injury. NASH is also associated with obesity yet improves with only modest weight loss. To distinguish the role of obesity versus sustained consumption of a trans-fat containing diet in causing NASH, mice with obesity and NASH induced by consuming a high trans-fat diet for 16 weeks were subsequently fed standard chow or maintained on trans-fat chow for another 8 weeks. The accumulation, partitioning and loss of trans-fats in the major hepatic lipid pools during and after trans-fat consumption were determined. Obese mice switched to standard chow remained obese but steatohepatitis improved. trans-fats were differentially incorporated into the major hepatic lipid pools and the loss of trans-fats after crossover to control chow was greatest in the cholesteryl ester pool. In summary, dietary changes can improve the biochemical and histopathological changes of NASH despite persistent obesity in mice. Analysis of hepatic lipids confirmed that dietary trans-fats accumulate in the major lipid pools and are released differentially with diet normalization. The substantial loss of trans-fats from the cholesteryl ester pool in parallel with improvement in NASH suggests that this pool of trans-fats could play a role in the pathogenesis of NASH.

Nonalcoholic fatty liver disease (NAFLD), which is characterized by accumulation of excessive amounts of fat in the liver, and nonalcoholic steatohepatitis (NASH), the subset of NAFLD associated with a significant risk of progression to cirrhosis and hepatocellular carcinoma, have become the most common forms of chronic liver disease. NASH is frequently associated with obesity, type 2 diabetes mellitus and other components of the metabolic syndrome but can occur without these risk factors. It is often suspected clinically by imaging evidence of excess fat in the liver and elevated serum aminotransferase levels, but a diagnosis of NASH can only be established by liver biopsy. The primary cause of hepatocellular injury in NASH is likely the generation of biologically active lipids generated from an increased flux of fatty acids through the liver. These fatty acids originate from de novo lipogenesis, or the synthesis of fatty acids from carbohydrates in the liver, and from release of fatty acids from adipose tissue, especially in the setting of insulin resistance. Lifestyle modification with a focus on healthy eating habits and sustained weight loss is the primary mode of treatment. Exercise, even without weight loss is beneficial. A number of drug therapies are currently being evaluated in clinical trials focused on resolution of NASH and improvement in fibrosis.

Background/Aims: In the past, nonalcoholic steatohepatitis has been described mostly in obese women with diabetes. The aim of this study was to describe a series of patients with nonalcoholic steatohepatitis with a different clinical profile. Methods: The clinical, biochemical, and histological features of 33 patients with nonalcoholic steatohepatitis seen from July 1990 to June 1993 were analyzed. Results: The mean age was 47 years. All patients were antibody to hepatitis C virusnegative. Nineteen of 33 (58%) were men, 20 of 33 (61%) were nonobese, 26 of 33 (79%) had normal glucose levels, and 26 of 33 (79%) had normal lipid levels. Fourteen of 33 (42%) had normal glucose and lipid levels and were not obese. Thirteen of 33 (39%) had pathological increases in fibrosis, 5 of whom had micronodular cirrhosis. Of these 13 with severe, progressive disease, 8 (62%) were women, 8 (62%) were obese, 4 (31%) were diabetic or had an elevated glucose level, and 3 (23%) had hyperlipidemia. Although serum iron studies (transferrin saturation and ferritin) were abnormal in 18 of 31 (58%), no patient had hemochromatosis. Conclusions: Nonalcoholic Steatohepatitis can be a severe, progressive liver disease leading to the development of cirrhosis. It should no longer be considered a disease predominantly seen in obese women with diabetes.

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (3H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon.

L-Buthionine-[S,R]-Sulfoximine (BSO) decreases glutathione levels in various organs by inhibition of gamma-glutamylcysteine synthetase. We have examined the levels of total glutathione and oxidized glutathione in the pancreas of mice, as well as serum amylase and pancreatic histology, after BSO administration in two different ways. The injection of a single dose of BSO (5 mmol/kg body wt) decreased total glutathione to 10% of the control value. A similar depletion was observed after 24 h of oral administration of a 10 mM BSO solution, without changes in the levels of oxidized glutathione. BSO-induced pancreatic glutathione depletion--even if maintained for up to 14 d--did not cause morphological alterations of the pancreas or hyperamylasemia. Thus pancreatic glutathione depletion in itself does not lead to pancreatitis, although during development of experimental acute pancreatitis, glutathione depletion has been described. BSO might be used in animal models to weaken the glutathione-based acinar defense mechanisms against oxidant stress or to alter other physiologic processes in which glutathione is involved.

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.

Data continue to emerge linking the gut-liver axis and nonalcoholic fatty liver disease (NAFLD). More than 500 bacterial species exist within the alimentary tract, the composition of which has been implicated in obesity and its downstream complications. Early animal studies showed that transplantation of gut microbial species from conventionally raised mice into germ-free mice led to increased fat storage and insulin resistance (1). In nonalcoholic steatohepatitis, small intestinal bacterial overgrowth and increased TNF-a production have also been observed. Elevated cytokine concentrations are thought to increase intestinal permeability via disruption of intracellular tight junctions, resulting in progressive inflammation and fibrosis within the liver (2, 3). Interestingly, the host’s interaction with the gut microbiome may be a 2-way street because recent studies have shown that the gut inflammatory response can also influence the components of the microbiota in mice (4). Modulating the gut microbiome via probiotics and prebiotics has been shown to be beneficial in NAFLD in both animal and human studies (5, 6). Probiotics, which are live commensal organisms, and prebiotics, which are nondigestible oligosaccharides and polysaccharides that can stimulate the growth of intestinal bacteria, can be used together in what is known as a synbiotic. A recent meta-analysis reported favorable results with the use of probiotics, specifically declines in concentrations of alanine aminotransferase, aspartate aminotransferase, and TNF-a and measures of insulin resistance (6). Of 4 randomized controlled trials conducted, 2 trials included the use of synbiotics by cotreatment with fructooligosaccharides (FOS), a prebiotic (7, 8). FOS can promote potentially beneficial growth of bifidobacteria, and a trial using FOS alone was reported to cause improvement in aminotransferases (9). Of note is that one of the previous trials in patients with nonalcoholic steatohepatitis included liver biopsies that showed improved liver histology after 6 mo of a synbiotic containing Bifidobacterium longum and FOS (7). In this issue of the Journal, Eslamparast et al (10) add to this evidence with the results of a study that used a synbiotic formulation in NAFLD in a double-blind, randomized, placebocontrolled trial. Fifty-two nondiabetic patients with newly diagnosed NAFLD by ultrasound and an alanine aminotransferase concentration .60 U/L were randomly assigned to receive a synbiotic formulation including Lactobacillus casei, Lactobacillus rhamnosus, Streptococcus thermophiles, Bifidobacterium breve, Lactobacillus acidophilus, B. longum, and Lactobacillus bulgaricus with FOS or placebo and were followed for 28 wk. Important findings of this study were improvements in aminotransferases as well as a decline in baseline mild fibrosis as estimated by FibroScan (echosens). Whereas this study did not include liver histology as an endpoint, the authors provided evidence that systemic inflammation decreased with a decline in circulating TNF-a concentrations and diminished nuclear transcription factor jB activation in circulating mononuclear leukocytes. These observations are consistent with previous findings with both probiotics and prebiotics (6). Whether these measures represent inflammation at the level of the gut, the liver, adipose tissue, or throughout the body remains to be determined. Without specific interventions to manipulate components of the inflammation (eg, anti–TNF-a therapies or other specific pharmacologic antiinflammatory approaches), which components of inflammation play a causal role and which are consequences of liver injury is yet to be sorted out. It is tempting to attribute causality to the inflammatory axis as the authors of this study did, but it may be premature to do so without confirmatory data. The use of FibroScan to measure liver stiffness in this trial yielded provocative data. There appeared to be a reduction in liver stiffness, but it is worth noting that both the preand posttreatment liver stiffness measures were near normal. Liver stiffness measures are also influenced by the degree of steatosis and other factors such as the fasting state of the subject (11). Without corresponding liver biopsies, it is difficult to know the meaning of improved liver stiffness values when the values are near the normal range. Although the number of subjects treated was relatively small, this study supports the safety of synbiotic use and corroborated the findings of previous trials with improvement in not only aminotransferases but also measures of fibrosis, cytokine concentrations, and insulin sensitivity. Future studies with long-term follow-up after stopping treatment to assess if the benefits of

Insulin sensitizers are attractive candidate therapies for nonalcoholic steatohepatitis mainly because of the strong association between this disease and insulin resistance. This review provides a critical overview of the mechanisms of action, clinical trial results, and safety issues of metformin and glitazones in nonalcoholic steatohepatitis. It also highlights methodological challenges for trial design and proposes endpoints for future proof of principle, registrational, and postmarketing trials.

A significant body of evidence now forces us to rethink the causes of NASH. Once thought to be a disease caused by triglyceride accumulation in hepatocytes with subsequent oxidant stress and lipid peroxidation causing inflammation and fibrosis, new data from animal studies and a limited number of human studies now provide convincing evidence that triglyceride accumulation does not cause insulin resistance or cellular injury in the liver. The lipotoxic liver injury hypothesis for the pathogenesis of NASH suggests that we need to focus our therapeutic efforts on reducing the burden of fatty acids going to the liver or being synthesized in the liver. This can be accomplished by improving insulin sensitivity at the level of adipose tissue to prevent inappropriate peripheral lipolysis and by preventing unnecessary de novo lipogenesis in the liver. Excess carbohydrates are the major substrates for de novo lipogenesis, and thus, reducing carbohydrate consumption through dietary changes and increasing muscle glucose uptake through exercise remain important cornerstones of treatment and prevention of lipotoxic liver injury, a disease hitherto called NASH.

Studies in animal models suggest that oxygen radicals may be important in the pathogenesis of acute pancreatitis. Because glutathione is an essential component of the defense against radical-mediated cellular injury, we investigated whether pancreatic glutathione content is influenced by inducing acute pancreatitis and whether augmenting the intracellular supply of glutathione would alter the course of pancreatitis. Caerulein, a decapeptide cholecystokinin analogue, induces acute necrotizing pancreatitis in mice when given in high doses (50 micrograms/kg per h) over a period of 6 h. The pancreatic glutathione content (total, GSH + GSSG) in mice treated with high-dose caerulein fell to 17% of normal within 4 h of beginning caerulein and recovered toward normal after discontinuing caerulein treatment. Mice treated with glutathione monoethyl ester (20 mmol/kg 1 h before caerulein, 10 mmol/kg 3 and 7 h after starting caerulein) were found to have blunted depletion of pancreatic glutathione, diminished histologic evidence of pancreatitis (necrosis, inflammation, and vacuolization), and lower serum amylase values compared with mice treated with caerulein alone. These findings suggest that the profound depletion of pancreatic glutathione caused by hyperstimulation of the pancreas with caerulein is critically important in the pathogenesis of acute caerulein-induced pancreatitis.

Adult nonalcoholic fatty liver disease (NAFLD) is characterized by absent or mild portal chronic inflammation (CI); in children, portal CI may be predominant. This study correlated clinical features with portal CI. Centrally-graded biopsies and temporally-related clinical parameters from 728 adults and 205 children. From the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) were evaluated. Mild, more than mild and no portal CI were found in 60%, 23% and 16% of adult biopsies and 76%, 14% and 10% of pediatric biopsies. Autoantibodies, and elevated alanine aminotransferase were not associated with portal CI. Clinical features associated with “more than mild” in adults were older age (P < 0.0001), female gender (P = 0.001), higher body mass index (P < 0.0001), elevated insulin levels (P = 0.001), higher homeostasis model assessment of insulin resistance score (HOMA-IR) (P < 0.0001), and medications used for NAFLD (P = 0.0004), diabetes (P < 0.0001), and hypertension (P < 0.0001). “More than mild” in the pediatric biopsies correlated with younger age (P = 0.01), but not with body mass index, insulin or HOMA-IR. In both groups, lobular and portal inflammation scores had no association, but there was an association with definite steatohepatitis (P < 0.0001). Features associated in the adult biopsies with “more than mild” were steatosis amount (P = 0.01) and location (P < 0.0001), ballooning (P < 0.0001), and advanced fibrosis (P < 0.0001). In the pediatric biopsies, “more than mild” was associated with steatosis location (P = 0.0008) and fibrosis score (P < 0.0001), specifically, the portal/periportal fibrosis or greater fibrosis) (P < 0.01). Conclusion: Increased portal CI is associated with many clinical and pathologic features of progressive NAFLD in both adults and children, but not with ALT, autoantibodies, or lobular inflammation. More than mild portal CI in liver biopsies of untreated NAFLD may be considered a marker of advanced disease. (HEPATOLOGY 2009.)

The aims of this study were to determine whether combining features of a western lifestyle in mice with trans fats in a high-fat diet, high-fructose corn syrup in the water, and interventions designed to promote sedentary behavior would cause the hepatic histopathological and metabolic abnormalities that characterize nonalcoholic steatohepatitis (NASH). Male C57BL/6 mice fed ad libitum high-fat chow containing trans fats (partially hydrogenated vegetable oil) and relevant amounts of a high-fructose corn syrup (HFCS) equivalent for 1–16 wk were compared with mice fed standard chow or mice with trans fats or HFCS omitted. Cage racks were removed from western diet mice to promote sedentary behavior. By 16 wk, trans fat-fed mice became obese and developed severe hepatic steatosis with associated necroinflammatory changes. Plasma alanine aminotransferase levels increased, as did liver TNF-α and procollagen mRNA, indicating an inflammatory and profibrogenic response to injury. Glucose intolerance and impaired fasting glucose developed within 2 and 4 wk, respectively. Plasma insulin, resistin, and leptin levels increased in a profile similar to that seen in patients with NASH. The individual components of this diet contributed to the phenotype independently; isocaloric replacement of trans fats with lard established that trans fats played a major role in promoting hepatic steatosis and injury, whereas inclusion of HFCS promoted food consumption, obesity, and impaired insulin sensitivity. Combining risk factors for the metabolic syndrome by feeding mice trans fats and HFCS induced histological features of NASH in the context of a metabolic profile similar to patients with this disease. Because dietary trans fats promoted liver steatosis and injury, their role in the epidemic of NASH needs further evaluation.

Clinical predictors of advanced non-alcoholic liver disease (NAFLD) are needed to guide diagnostic evaluation and treatment.To better understand the demographics of NAFLD and risk factors for advanced disease, this study analysed 827 patients with NAFLD at two geographically separate tertiary medical centres.The cohort was 51% female and had a median body mass index (BMI) of 33 kg/m(2); 3% had a normal BMI. Common co-morbidities included hypertension (60%) and diabetes (35%); insulin resistance was present in 91% and advanced fibrosis in 24% of patients. When comparing patients with no fibrosis or mild fibrosis to those with advanced fibrosis, BMIor = 28 kg/m(2), age50 years, and aspartate transaminase/alanine aminotransferase (AST/ALT) ratioor = 0.8, a quantitative assessment check index (QUICKI) score0.294 (equivalent to homeostasis model assessment (HOMA)6.2) and the presence of diabetes mellitus (DM) were individually associated by univariate analysis with odds ratios (ORs) ofor = 2.4 for advanced fibrosis. Based on the results of forced entry logistic regression analysis, three variables were combined in a weighted sum (BMIor = 28 = 1 point, AAR ofor = 0.8 = 2 points, DM = 1 point) to form an easily calculated composite score for predicting advanced fibrosis called the BARD score. A score of 2-4 was associated with an OR for advanced fibrosis of 17 (confidence interval 9.2 to 31.9) and a negative predictive value of 96%.Insulin resistance and its co-morbidities are often present in patients with NAFLD. An easily calculated score based on readily available clinical data can reliably exclude the presence of advanced fibrosis in these patients, particularly among non-diabetics.

was identified in 30% of subjects. In this group, SBMI and LBMI were strongly correlated with HVPG (r=0.56, p=0.006 and r=0.58, p=0.002 respectively), but not with AST to platelet index (APRI) or FIB-4 scores (r=0.14, p=0.49 and r=0.30, p= 0.15 respectively). In those with viral hepatitis, SBMI and LBMI were not correlated with HVPG values (r=0.16, p=0. 56 and r=0.04, p=0.89), while FIB-4 correlated with HVPG (r=0.71, p=0.002) and APRI trended towards significance (r=0.49, p=0.056).Conclusions: As expected in viral hepatitis, HVPG and non-invasive scoring systems correlated, however spleen and liver volumes did not correlate with HVPG. In NCPH spleen and liver volumes strongly correlated with HVPG, however non-invasive scoring systems did not correlate with HVPG. The finding that spleen and liver volumes may serve as a non-invasive surrogate for portal hypertension in NCPH should be further explored.

of cirrhosis is highly desirable because it simplifies post-LT management. Material and Methods: In this adaptive open-label, prospective randomized phase III North American clinical trial (Study 988, NCT01804829) conducted in 2 parts, any antiviral therapy (AVT) pre-LT (resulting in RNA

Ingestion of wild mushrooms has led to unintentional poisonings caused by mistaken identity. We report 3 cases of exposure to Amanita bisporigera, demonstrating dose-related toxicity. The use of nasobiliary drainage as a novel approach to interrupting the enterohepatic circulation of amatoxins is illustrated. Pathophysiology and treatment of Amanita poisoning are reviewed.

OBJECTIVES: The clinical significance of portal hypertensive gastropathy (PHG) in patients with compensated liver disease is not well established. The aim of this study was to determine the prevalence and correlates of PHG in a large cohort of patients with chronic hepatitis C virus (HCV) infection and bridging fibrosis/compensated cirrhosis entering the randomized phase of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial (HALT-C). METHODS: The presence and severity of PHG in 1,016 HCV patients with no prior history of gastrointestinal bleeding was determined at surveillance endoscopy using the New Italian Endoscopy Club criteria. RESULTS: Overall, 37% of HALT-C patients had PHG with 34% having mild and 3% with severe changes. The mucosal mosaic pattern was identified in 33%, red marks in 15%, and gastric antral vascular ectasia (GAVE) features in only 3%. Independent correlates of PHG included biochemical markers of liver disease severity (lower serum albumin, higher bilirubin), portal hypertension (lower platelet count), insulin resistance (higher glucose), and non-African American race. Independent correlates of GAVE included a history of smoking, nonsteroidal anti-inflammatory drugs (NSAIDs) use within the past year, and higher serum bilirubin and glucose levels. There was a strong positive association between the presence of PHG and esophageal varices ( p < 0.0001). CONCLUSIONS: PHG is associated with the histological and biochemical severity of liver disease in patients with HCV and advanced fibrosis but is mild in most patients. The clinical relevance of these findings will be further explored during the randomized phase of the HALT-C study.

Relatively recently, the liver has been recognized as a major target of injury in patients with insulin resistance or the metabolic syndrome. Insulin resistance is associated with fat accumulation in the liver, a condition called nonalcoholic fatty liver disease (NAFLD). Excess fat in the liver is not a benign condition. Some patients with NAFLD develop necroinflammatory changes in the liver called nonalcoholic steatohepatitis (NASH) and a fraction of those will develop cirrhosis. About 20% all adults have NAFLD and 2% to 3% of adults have NASH. Approximately 20% of patients with NASH are at risk for developing cirrhosis and subsequently dying from end-stage liver disease. The diagnosis of NASH requires a high index of suspicion, especially in obese patients over the age of 45 years who have diabetes, because these are the patients at greatest risk for developing cirrhosis. Treatment focuses on addressing the underlying insulin resistance with increased exercise and weight reduction.

Thirty overweight patients with clinically characterized and biopsy proven nonalcoholic steatohepatitis (NASH) were enrolled in a 48-week treatment trial with rosiglitazone, a peroxisome proliferator-activator receptor (PPAR)-gamma agonist that enhances insulin sensitivity. Improvement in laboratory liver tests, insulin resistance and liver fat content were documented; blinded biopsy review demonstrated decreases in necroinflammatory activity or grade and in individual components of grade, and changes in the relationship of lobular and portal inflammation as well as in the nature of perisinusoidal fibrosis. The current study identified correlations of histological features of the protocol entry biopsy specimens with contemporaneous laboratory and imaging tests. Significant correlations with histologically assessed steatosis were liver fat, evaluated by computed tomography (P = 0.001); mean HbA1C, a measure of glycemic control (P = 0.004); and QUICKI, a measure of insulin sensitivity (P = 0.05). Histologically determined grades of steatohepatitis (SH) correlated with HbA1C (P = 0.01), and a trend toward elevated fasting glucose levels was seen. No subject in the study was cirrhotic at entry; fibrosis scores of the 30 subjects did not significantly correlate with age, gender, body mass index, or clinical tests. All subjects underwent 3 biopsies (prior, entry, and posttreatment), and all had undergone a prior biopsy with diagnostic SH. By blinded analysis, 7 study entry biopsy specimens did not fulfill published strict criteria for SH. Laboratory results from these subjects included normal fasting glucose level and, compared with the 23 subjects with criteria for SH, lower mean alanine aminotransferase and aspartate aminotransferase levels (P = 0.02 for both), less insulin resistance (P = 0.03), and lower mean HbA1C (P = 0.001). We conclude that biopsy findings determined by blinded analysis correlated with image-detected steatosis, laboratory markers of hepatic inflammation, insulin resistance, and long-term glycemia; the findings confirm the usefulness of strict histological criteria in the evaluation of NASH.

A mouse model using repetitive acinar cell injury caused by supraphysiologic doses of cerulein to induce the characteristic fibrosis and loss of acinar cell mass found in human chronic pancreatitis was employed to identify early changes in gene expression. A gene array was used to detect changes in 18,000 expressed sequence tags; changes in specific transcripts were confirmed by RNase protection assays. These methods identified SPINK3, the mouse homologue of human and rat protease inhibitor genes, as being highly expressed in the pancreas and induced after pancreatic injury. Because SPINK3 may be an important serine protease inhibitor, its up-regulation may reflect an important endogenous cytoprotective mechanism in preventing further injury. The up-regulation of SPINK3 was specific; the mouse homologue of the zymogen-processing protein ZG-16p was also highly expressed in the pancreas but sharply down-regulated early in the course of injury. These findings suggest that the pancreatic acinar cell may respond to injury with a program of self-preservation and loss of normal function.

Insulin resistance (IR) commonly is associated with nonalcoholic steatohepatitis (NASH). To establish whether IR causes NASH, this study was undertaken to determine if improving IR would improve the histologic features that define NASH. Thirty adults with prior biopsy evidence of NASH were enrolled to receive rosiglitazone, 4 mg twice daily for 48 weeks. All patients were overweight (body mass index [BMI] > 25 kg/m(2)) and 23% were severely obese (BMI > 35 kg/m(2)); 50% had impaired glucose tolerance or diabetes. Liver biopsy specimens were obtained before beginning treatment and at treatment completion. Twenty-six patients had posttreatment biopsies; of these, 22 had initial protocol liver biopsies that met published criteria for NASH on subsequent blinded evaluation. Within this initial NASH group, the mean global necroinflammatory score significantly improved with treatment and biopsies of 10 patients (45%) no longer met published criteria for NASH after treatment. Significant improvement in hepatocellular ballooning and zone 3 perisinusoidal fibrosis also occurred. Five patients withdrew early; the 25 patients completing 48 weeks of treatment had significantly improved insulin sensitivity and mean serum alanine aminotransferase (ALT) levels (104 initially, 42 U/L at the end of treatment). Adverse effects led to withdrawal of 3 patients (10%). Weight gain occurred in 67% of patients and the median weight increase was 7.3%. Within 6 months of completing treatment, liver enzyme levels had increased to near pretreatment levels. In conclusion, improving insulin sensitivity with rosiglitazone resulted in improved histologic markers of NASH, an observation suggesting that insulin resistance contributes to its development and that improving insulin sensitivity may be important in treating this liver disease.

Hepatotoxicity caused by the mushroom poison alpha-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of alpha-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for alpha-amanitin uptake, we tested the hypothesis that Na(+)-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular alpha-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K(m) and V(max). A toxicologically relevant functional assay for alpha-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1beta (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 micro M alpha-amanitin for 6-10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P0.02 compared with stimulated transfected cells without alpha-amanitin). These findings demonstrate that Ntcp may be an important mediator of alpha-amanitin uptake by the liver.

Fatty liver disease that develops in the absence of alcohol abuse is recognized increasingly as a major health burden. This report summarizes the presentations and discussions at a Single Topic Conference held September 20-22, 2002, and sponsored by the American Association for the Study of Liver Diseases. The conference focused on fatty liver disorders. Estimates based on imaging and autopsy studies suggest that about 20% to 30% of adults in the United States and other Western countries have excess fat accumulation in the liver. About 10% of these individuals, or fully 2% to 3% of adults, are estimated to meet current diagnostic criteria for nonalcoholic steatohepatitis (NASH). Sustained liver injury leads to progressive fibrosis and cirrhosis in a fraction, possibly up to one third, of those with NASH, and NASH may be a cause of cryptogenic cirrhosis. NASH is now a significant health issue for obese children as well, leading to cirrhosis in some. The diagnostic criteria for NASH continue to evolve and rely on the histologic findings of steatosis, hepatocellular injury (ballooning, Mallory bodies), and the pattern of fibrosis. Generally recognized indications for biopsy include establishing the diagnosis and staging of the injury, but strict guidelines do not exist. Liver enzymes are insensitive and cannot be used reliably to confirm the diagnosis or stage the extent of fibrosis. Older age, obesity, and diabetes are predictive of fibrosis. The pathogenesis of NASH is multifactorial. Insulin resistance may be an important factor in the accumulation of hepatocellular fat, whereas excess intracellular fatty acids, oxidant stress, adenosine triphosphate (ATP) depletion, and mitochondrial dysfunction may be important causes of hepatocellular injury in the steatotic liver. Efforts are underway to refine the role of insulin resistance in NASH and determine whether improving insulin sensitivity pharmacologically is an effective treatment. An altered lifestyle may be a more effective means of improving insulin sensitivity. The research agenda for the future includes establishing the role of insulin resistance and abnormal lipoprotein metabolism in NASH, determining the pathogenesis of cellular injury, defining predisposing genetic abnormalities, identifying better noninvasive predictors of disease, and defining effective therapy.

Hyperinsulinemia may cause hepatic steatosis and non-alcoholic steatohepatitis (NASH). The aims of this pilot study were to examine the safety of using the insulin-sensitizing peroxisomal proliferator activated receptor (PPAR) gamma ligand rosiglitazone in patients with NASH and determine whether improved insulin sensitivity correlates with improved fatty liver.Thirty subjects with NASH and elevated alanine aminotransferase (ALT) levels received rosiglitazone, 4 mg twice daily for 48 weeks; the preliminary results presented here were obtained at 24 weeks. Insulin sensitivity was measured using fasting insulin and glucose levels and liver fat content was estimated by CT imaging.By 24 weeks, rosiglitazone improved insulin sensitivity and reduced liver fat content. The mean ALT decreased from 86 to 37 U/l (P0.01). Four subjects (13%) withdrew, one because of a rise in ALT from 59 to 277 U/l that coincided with concomitant prednisone use. Subjects experienced a mean weight gain of 3.5% and hemoglobin drop of 1.1 g/dl.Treatment of NASH with rosiglitazone for 24 weeks improved insulin sensitivity, reduced liver fat content and improved biochemical evidence of hepatocellular injury. These preliminary data provide evidence that hyperinsulinemia may be a cause of NASH. Strategies to improve insulin sensitivity as a treatment of NASH deserve further investigation.

In this special issue of Journal of Lipids, we acknowledge the contributions by several experts offering timely perspectives on the complex interactions between bile acids and nuclear receptors (NRs) on lipid metabolism and liver diseases at different levels and contexts in the body. NRs are found within the interior of cells and are defined as ligand-activated transcriptional regulators of several key aspects of body physiology and pathophysiology. NRs regulate gene transcription through interaction with cellular coactivators and corepressors. In the liver, NRs play a key role in a large variety of metabolic processes such as cholesterol, bile acid, fatty acid, and glucose homeostasis, as well as drug disposition. Also, additional critical processes involving the pathophysiology of liver diseases—inflammation and fibrosis, regeneration, cell differentiation, and tumor formation—are modulated by NRs. Of note, NRs are or might soon become drug targets. Despite the huge accumulation of knowledge in the field, the true comprehension of interactions between bile acids and NRs on lipid metabolism and hepatobiliary diseases has remained elusive. Thus continuous efforts are being made to understand the molecular functions of NRs, the significance of bile acid-controlled signaling pathways, and interactions of NRs on a number of metabolic and hepatic diseases. The paper of T. Li and Y. L. Chiang is focused on the role of bile acid signaling in the regulation of glucose and lipid metabolism. Besides their detergent properties and key physiological functions, bile acids are also acting as potent metabolic regulators of glucose and lipid homeostasis. The identification of bile acid-activated nuclear receptor farnesoid X receptor (FXR) and cell surface G-protein-coupled receptor TGR5 has significantly advanced our understanding on how bile acid signaling regulates cellular metabolism in health and disease. Thus, novel therapeutic strategies can be envisioned which target bile acid metabolism for the treatment of metabolic disorders such as obesity, insulin resistance, and the metabolic syndrome. NRs comprise one of the most abundant classes of transcriptional regulators of metabolic diseases and have emerged as promising pharmaceutical targets. The paper by G. Garruti et al. deals with the myriad roles of small heterodimer partner (SHP), a unique orphan nuclear receptor lacking a DNA-binding domain, but containing a putative ligand-binding domain. About half of mammalian NRs and several transcriptional coregulators can interact with SHP. SHP is a transcriptional regulator affecting multiple key biological functions and metabolic processes including cholesterol, bile acid, and fatty acid metabolism, as well as reproductive biology and glucose-energy homeostasis. In humans, studies are emerging on the association of SHP genetic variation with birth weight, high body mass index, obesity, insulin resistance, and diabetes. Future research must be focused on synthetic ligands acting on SHP as a potential therapeutic target in a series of metabolic abnormalities. One important issue in lipidology is the understanding of the molecular mechanisms whereby cholesterol and fatty acids are absorbed from the intestine and are transported to the liver. The cholesterol absorption inhibitor ezetimibe can significantly reduce plasma total and LDL 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. The paper by O. de Bari et al. emphasizes the novel concept that, ezetimibe treatment also induces a complete resistance to two frequent metabolic abnormalities, namely, cholesterol gallstones and nonalcoholic fatty liver disease (NAFLD). Furthermore, it prevented hypercholesterolemia in mice on a Western diet. This model has high translational value and points to a key role for chylomicrons, the natural lipid carriers used by enterocytes to transport cholesterol and fatty acids into the body. The hypothesis that ezetimibe could prevent two prevalent hepatobiliary diseases (i.e., cholesterol cholelithiasis and liver steatosis) possibly through the regulation of chylomicron-derived cholesterol and fatty acid metabolism in the liver is discussed here. Because several proteins are implicated in determining biliary lipid secretion in the liver and are regulated by several transcription factors, including nuclear receptors liver X receptor (LXR) and FXR, the paper by M. C. Vazquez et al. is focused on molecular mechanisms underlying the link between nuclear receptor function and the formation of cholesterol gallstones. A potent role for estrogen receptors in the pathogenesis of cholesterol gallstone disease, involving both genomic and nongenomic activation of signaling pathways, is discussed. Evidence in this respect is heavily supported by human and murine genetic, physiological, pathophysiological, and pharmacological studies. Indeed, expanding the knowledge about the role of NRs in gallstone formation will certainly lead to the discovery of novel and more effective therapeutic strategies in a typical example of a metabolic “mass disease,” that is, cholesterol cholelithiasis. In the wide field of lipopathy, NAFLD is currently evolving as the most common liver disease worldwide, with potential costly and severe sequelae, including liver cirrhosis and hepatocellular carcinoma. In his paper, M. Fuchs underscored the concept that NAFLD not only represents an insulin resistance state characterized by a cluster of dysmetabolic cardiovascular risk factors, but also represents an independent risk factor for cardiovascular diseases. Of note, the bile acid-activated nuclear receptor FXR has been shown to play a role not only in bile acid but also in lipid (cholesterol and triglyceride) metabolism and glucose homeostasis. Specific targeting of FXR may be an elegant and very effective way to readjust dysregulated nuclear receptor-mediated metabolic pathways. Activation of FXR may result in not only beneficial actions but also potential undesirable side effects. One example is the (still unpredictable) balance between pro- and anti-atherogenic effects of FXR activation. J. A. Lopez-Velazquez et al. described the important role of several NRs in the liver as regulators of several critical metabolic steps involved in the pathogenesis of NAFLD. Such crucial steps include fat storage, export, uptake, oxidation, and lipolysis. A whole family of NRs is targeted by many ligands controlling lipid metabolism including fatty acids, oxysterols, and lipophilic molecules. Understanding the molecular mechanisms underlying the involvement of NRs in the pathogenesis of NAFLD may, therefore, offer targets for the development of new treatments of one of the most frequent chronic liver diseases worldwide. In their paper, R. Mullenbach et al. provided an update on genetic variants of NRs involved in regulating important aspects of liver metabolism. One such aspect is the application of NRs in genetic diagnosis of monogenic (Mendelian) liver diseases and their uses in clinical diagnosis. Moreover, a role of NR polymorphisms in common diseases can be anticipated, linking regulatory networks to complex and variable phenotypes. Technical advances contribute to the restless expansion of knowledge and include transgenic animal models, expression quantitative trait loci (eQTL) mapping, and genomewide association studies (GWASs). Thus, it is highly likely that personal genome information might eventually be able to predict a variety of risks associated with an individual's lifestyle such as high fat diet and alcohol as well as susceptibility to infectious liver diseases such as hepatitis B or C. Menopause is a consequence of the normal aging process in women and it is thought that menopause is associated with a higher risk for cardiovascular diseases. Indeed, the post-menopause lipid profile is often altered, which represents a risk factor for cardiovascular diseases. The paper by P. J. Oliveira et al. reports on the mechanisms linking alterations of mitochondrial bioenergetics in the heart, as a consequence from normal aging and/or from the menopausal process, to decreased fatty acid oxidation and accumulation of fatty acid intermediates in the cardiomyocyte cytosol. Such lipotoxic consequences might represent the important link to increased cardiovascular risk in the menopausal women. In conclusion, the field of lipidology has become even more complex and exciting when considering that the discovery of NRs and their pleiotropic functions have opened the way to multidimensional, multidisciplinary and translational studies. Since NRs are involved in virtually all physiological functions, understanding how NRs work is therefore essential to explain the complex pathophysiological mechanisms underlying liver and extrahepatic diseases. A new era in which NRs will represent valid therapeutic targets for several disorders is hopefully approaching. Lastly, we hope that this contribution will also help both young and experienced investigators in their daily difficult task to expand their research in the field of experimental and clinical lipidology in health and disease. David Q.-H. Wang Brent A. Neuschwander-Tetri Piero Portincasa William M. Pandak

Cross-sectional studies have associated serum levels of the keratin 18 (K18) fragment with histologic features of liver in individuals with nonalcoholic fatty liver disease (NAFLD). We investigated the relationship between changes in serum levels of K18 and changes in liver histology in adults and children with NAFLD.We measured levels of K18 in stored serum samples collected at baseline and various time points from 231 adults with nonalcoholic steatohepatitis and 152 children with NAFLD who participated in 2 separate prospective randomized clinical trials. Liver biopsy specimens collected at baseline and week 96 were reviewed centrally.There were greater decreases in serum levels of K18 in adults with histologic improvement at week 96 than in those without histologic improvement at week 16 (decrease, 193 ± 293 vs 139 ± 467 U/L; P.001), week 48 (decrease, 232 ± 360 vs 113 ± 425 U/L; P.001), or week 96 (decrease, 269 ± 368 vs 97 ± 400 U/L; P.001). There were greater decreases in serum levels of K18 in children with histologic improvements than in those without histologic improvements at week 48 (decrease, 197 ± 467 vs 47 ± 350 U/L; P = .005) and week 96 (decrease, 206 ± 432 vs 2 ± 474 U/L; P.001). However, reductions in serum levels of K18 were not better than reductions in levels of alanine aminotransferase in identifying adults with histologic improvement (area under the receiver operator characteristic [AUROC], 0.71; 95% confidence interval [CI], 0.63-0.80; vs AUROC, 0.68; 95% CI, 0.61-0.79; P = .34) or children (AUROC, 0.72; 95% CI, 0.63-0.81; vs AUROC, 0.79; 95% CI, 0.70-0.87; P = .42).Decreases in serum levels of K18 are associated strongly with improved liver histologies in adults or children with NAFLD. However, reductions in K18 do not perform better than those in alanine aminotransferase level in identifying histologic changes in NAFLD.

of fibrosis in an elderly population using a non-invasive measure. There is a high prevalence of advanced fibrosis within an elderly population. Moreover, these data are the first to show the relationship between the FLI and NFS in a population with NAFLD. The significant correlation between FLI and NFS suggest a liver biopsy may not be necessary in this high risk population to determine the presence of significant fibrosis. 1Koehler E et al. External Validation of the Fatty Liver Index for Identifying Non-alcoholic Fatty Liver Disease in a Population-based Study. Clin Gastroenterol Hepatol. 2013; 11:1201-1204 2Angulo P et al. The NAFLD fibrosis score: a non-invasive system that identifies liver fibrosis in patients with NAFLD. Hepatology. 2007(Apr);45(4):846-54

Background: ApoA5, the newest member of the apolipoprotein family, is expressed exclusively in the liver. Although plasma ApoA5 concentrations in humans are very low (114258 ng/mL) and ~1,000-fold lower than those of ApoB100 on a molar basis, human and mouse studies have clearly shown a key role of ApoA5 in plasma triglyceride (TG) homeostasis. Because ~20% of ApoA5 is secreted into the circulation and its concentration is 7-fold higher in the liver than in plasma, we hypothesize that ApoA5 plays a critical role in regulating hepatic TG metabolism and ApoA5 deficiency profoundly perturbs the balance of daily input and output of fatty acids (FA) in the liver. Methods: Male ApoA5 KO and WT mice were fed a chow diet for 12 mo. Liver pathology was examined by H&E and Sirius Red staining. Lipid droplets (LD) and endoplasmic reticulum (ER) and Golgi fractions were isolated from homogenized liver by ultracentrifugation. Gene expression and protein content were studied by qRT-PCR and Western blots. Lipidomic analysis of liver was performed by ESI-MS/MS. Results: During the 12-mo period, on chow, ApoA5 deficiency significantly reduced TG disposal by inhibiting FA β-oxidation through repressing carnitine palmitoyltransferase-1, coupled with reduced energy expenditure. In WT mice, because it is a LD-associated protein, ApoA5 guided TG-rich LD to the ER for VLDL assembly and subsequently to the Golgi apparatus for hepatic VLDL secretion through its interaction with Plin2, Cideb and ApoB100. ApoA5 deficiency reduces hepatic VLDL secretion by impairing its lipidation and maturation. As a result, accumulation of excess TG within the cytoplasm of hepatocytes induced the formation of numerous large LD, leading to liver steatosis. ApoA5 deficiency significantly increased de novo synthesis of ceramides, the major FAmetabolites, by enhancing hepatic serine palmitoyltransferase activity. Due to their hepatocellular lipotoxicity, increased ceramides induced hepatic lipoapoptosis through TNFα by stimulating both caspase-induced apoptosis and ROS-mediated mitochondrial dysfunction in ApoA5 KO, but not WT mice. These alterations triggered NASH with a main histological feature of hepatocellular ballooning in ApoA5 KO mice. Ceramide-induced hepatocyte apoptosis and activation of hepatic stellate cells promoted fibrogenesis by triggering a profibrogenic gene program, leading to liver fibrosis. Conclusions: ApoA5 deficiency in chow-fed mice over a long period leads to spontaneous development of NAFLD, gradually evolving from simple steatosis to NASH, and subsequently to liver fibrosis, which closely recapitulates many aspects of the pathogenic process of NAFLD in patients. These innovative studies will help us understand the mechanisms underlying the critical role of ApoA5 in the pathogenesis of NAFLD and provide novel targets to combat NAFLD by pharmacological intervention.

Insulin sensitizing agents, such as the thiazolidinediones pioglitazone and rosiglitazone, have been used in clinical trials for the treatment of nonalcoholic steatohepatitis, a common form of liver disease that is associated with insulin resistance. The results of these trials have been variable, suggesting that a deeper understanding of other contributing factors to the development of nonalcoholic steatohepatitis is needed to establish whether improving adipose insulin sensitivity might be important.

The cytochrome CYP3A gene products, expressed in mammalian liver, are essential for the metabolism of lipophilic substrates, including endogenous steroid hormones and prescription drugs. CYP3A enzymes are extremely versatile and are inducible by many of their natural and xenobiotic substrates. Consequently, they form the molecular basis for many clinical drug-drug interactions. The induction of CYP3A enzymes is species-specific, and we have postulated that it involves one or more cellular factors, or receptor-like xeno-sensors. Here we identify one such factor unequivocally as the nuclear receptor pregnenolone X receptor (PXR) and its human homologue, steroid and xenobiotic receptor (SXR). We show that targeted disruption of the mouse PXR gene abolishes induction of CYP3A by prototypic inducers such as dexamethasone or pregnenolone-16alpha-carbonitrile. In transgenic mice, an activated form of SXR causes constitutive upregulation of CYP3A gene expression and enhanced protection against toxic xenobiotic compounds. Furthermore, we show that the species origin of the receptor, rather than the promoter structure of CYP3A genes, dictates the species-specific pattern of CYP3A inducibility. Thus, we can generate 'humanized' transgenic mice that are responsive to human-specific inducers such as the antibiotic rifampicin. We conclude that SXR/PXR genes encode the primary species-specific xeno-sensors that mediate the adaptive hepatic response, and may represent the critical biochemical mechanism of human xenoprotection.

OJECTIVE: Steatohepatitis is a morphological pattern of liver injury that may be seen in alcoholic or nonalcoholic liver disease. This pattern may occur with obesity, diabetes, the use of certain drugs, or the cause may be idiopathic. The well-recognized histopathological features of nonalcoholic steatohepatitis (NASH) include hepatocellular steatosis and ballooning, mixed acute and chronic lobular inflammation, and zone 3 perisinusoidal fibrosis. Currently, there are no systems for grading necroinflammatory activity or for staging fibrosis as exist for various other forms of chronic liver disease. The purpose of this study was to develop such a grading and staging system and was based on review of liver biopsies from 51 patients with nonalcoholic steatohepatitis from Saint Louis University Health Sciences Center. METHODS: For determination of grade, 10 histological variables of activity were initially analyzed; an overall impression of mild, moderate, and severe was made and the variables considered to be most significant were used to develop the necroinflammatory grade. RESULTS: The histological lesions considered to be significant were: steatosis, ballooning, and intra-acinar and portal inflammation. A staging score was developed to reflect both location and extent of fibrosis. The fibrosis score was derived from the extent of zone 3 perisinusoidal fibrosis with possible additional portal/periportal fibrosis and architectural remodeling. Fibrosis stages are as follows: Stage 1, zone 3 perisinusoidal fibrosis; Stage 2, as above with portal fibrosis; Stage 3, as above with bridging fibrosis; and Stage 4, cirrhosis. CONCLUSION: We propose a grading and staging system that reflects the unique histological features of nonalcoholic steatohepatitis.

The recently identified hepatitis G virus (HGV, also named GB virus-C, GBV-C) appears to have similarities to hepatitis C virus and other flaviviridae. To better understand its clinical significance and hepatotropism, we collected liver tissue and matched serum samples from 56 patients undergoing liver transplantation. HGV/GBV-C RNA was detected by reverse transcription-nested PCR, using primers from the relatively conserved 5' noncoding region of the genome to detect HGV/GBV-C RNA and the amount was semiquantitatively estimated by serial 10-fold endpoint dilution. The presence and amount of HCV RNA was estimated by the same methodology. Seventeen patients (30%) had HGV/GBV-C RNA detectable either in liver or in serum, including two of three with cryptogenic liver disease. Interestingly, 5 of 17 (29%) patients had HGV/GBV-C RNA in serum but not liver, even with repeated testing of hepatic RNA from different portions of the liver. Furthermore, the titer of HGV/GBV-C RNA was significantly lower in liver than in serum in most samples (mean log titer, 1.33 vs. 2.56, P0.05). In contrast, all 21 patients with HCV RNA in serum also had the virus detectable in liver. In five patients coinfected with HCV and HGV/GBV-C, the mean titer of HCV RNA in liver was higher than that in serum (log titer, 2.8 vs. 3.0, P0.05). Thus, our results suggest that HGV/GBV-C is probably not hepatotropic and may replicate predominantly in sites other than the liver. These findings brings into question the role of HGV in causing significant liver disease.