Manal F. Abdelmalek

Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial

BACKGROUND The bile acid derivative 6-ethylchenodeoxycholic acid (obeticholic acid) is a potent activator of the farnesoid X nuclear receptor that reduces liver fat and fibrosis in animal models of fatty liver disease. We assessed the efficacy of obeticholic acid in adult patients with non-alcoholic steatohepatitis. METHODS We did a multicentre, double-blind, placebo-controlled, parallel group, randomised clinical trial at medical centres in the USA in patients with non-cirrhotic, non-alcoholic steatohepatitis to assess treatment with obeticholic acid given orally (25 mg daily) or placebo for 72 weeks. Patients were randomly assigned 1:1 using a computer-generated, centrally administered procedure, stratified by clinical centre and diabetes status. The primary outcome measure was improvement in centrally scored liver histology defined as a decrease in non-alcoholic fatty liver disease activity score by at least 2 points without worsening of fibrosis from baseline to the end of treatment. A planned interim analysis of change in alanine aminotransferase at 24 weeks undertaken before end-of-treatment (72 weeks) biopsies supported the decision to continue the trial (relative change in alanine aminotransferase -24%, 95% CI -45 to -3). A planned interim analysis of the primary outcome showed improved efficacy of obeticholic acid (p=0·0024) and supported a decision not to do end-of-treatment biopsies and end treatment early in 64 patients, but to continue the trial to obtain the 24-week post-treatment measures. Analyses were done by intention-to-treat. This trial was registered with ClinicalTrials.gov, number NCT01265498. FINDINGS Between March 16, 2011, and Dec 3, 2012, 141 patients were randomly assigned to receive obeticholic acid and 142 to placebo. 50 (45%) of 110 patients in the obeticholic acid group who were meant to have biopsies at baseline and 72 weeks had improved liver histology compared with 23 (21%) of 109 such patients in the placebo group (relative risk 1·9, 95% CI 1·3 to 2·8; p=0·0002). 33 (23%) of 141 patients in the obeticholic acid developed pruritus compared with nine (6%) of 142 in the placebo group. INTERPRETATION Obeticholic acid improved the histological features of non-alcoholic steatohepatitis, but its long-term benefits and safety need further clarification. FUNDING National Institute of Diabetes and Digestive and Kidney Diseases, Intercept Pharmaceuticals.

Fructose consumption as a risk factor for non-alcoholic fatty liver disease

BACKGROUND/AIMS While the rise in non-alcoholic fatty liver disease (NAFLD) parallels the increase in obesity and diabetes, a significant increase in dietary fructose consumption in industrialized countries has also occurred. The increased consumption of high fructose corn syrup, primarily in the form of soft drinks, is linked with complications of the insulin resistance syndrome. Furthermore, the hepatic metabolism of fructose favors de novo lipogenesis and ATP depletion. We hypothesize that increased fructose consumption contributes to the development of NAFLD. METHODS A dietary history and paired serum and liver tissue were obtained from patients with evidence of biopsy-proven NAFLD (n=49) without cirrhosis and controls (n=24) matched for gender, age (+/-5 years), and body mass index (+/-3 points). RESULTS Consumption of fructose in patients with NAFLD was nearly 2- to 3-fold higher than controls [365 kcal vs 170 kcal (p<0.05)]. In patients with NAFLD (n=6), hepatic mRNA expression of fructokinase (KHK), an important enzyme for fructose metabolism, and fatty acid synthase, an important enzyme for lipogenesis were increased (p=0.04 and p=0.02, respectively). In an AML hepatocyte cell line, fructose resulted in dose-dependent increase in KHK protein and activity. CONCLUSIONS The pathogenic mechanism underlying the development of NAFLD may be associated with excessive dietary fructose consumption.

Increased fructose consumption is associated with fibrosis severity in patients with nonalcoholic fatty liver disease

The rising incidence of obesity and diabetes coincides with a marked increase in fructose consumption. Fructose consumption is higher in individuals with nonalcoholic fatty liver disease (NAFLD) than in age‐matched and body mass index (BMI)‐matched controls. Because fructose elicits metabolic perturbations that may be hepatotoxic, we investigated the relationship between fructose consumption and disease severity in NAFLD. We studied 427 adults enrolled in the NASH Clinical Research Network for whom Block food questionnaire data were collected within 3 months of a liver biopsy. Fructose consumption was estimated based on reporting (frequency × amount) of Kool‐aid, fruit juices, and nondietary soda intake, expressed as servings per week, and classified into none, minimum to moderate (<7 servings/week), and daily (≥7 servings/week). The association of fructose intake with metabolic and histological features of NAFLD was analyzed using multiple linear and ordinal logistic regression analyses with and without controlling for other confounding factors. Increased fructose consumption was univariately associated with decreased age (P < 0.0001), male sex (P < 0.0001), hypertriglyceridemia (P < 0.04), low high‐density lipoprotein (HDL) cholesterol (<0.0001), decreased serum glucose (P < 0.001), increased calorie intake (P < 0.0001), and hyperuricemia (P < 0.0001). After controlling for age, sex, BMI, and total calorie intake, daily fructose consumption was associated with lower steatosis grade and higher fibrosis stage (P < 0.05 for each). In older adults (age ≥ 48 years), daily fructose consumption was associated with increased hepatic inflammation (P < 0.05) and hepatocyte ballooning (P = 0.05). Conclusion: In patients with NAFLD, daily fructose ingestion is associated with reduced hepatic steatosis but increased fibrosis. These results identify a readily modifiable environmental risk factor that may ameliorate disease progression in patients with NAFLD. HEPATOLOGY 2010

Betaine, a promising new agent for patients with nonalcoholic steatohepatitis: results of a pilot study

Betaine, a promising new agent for patients with nonalcoholic steatohepatitis: results of a pilot study

Noninvasive evaluation of hepatic fibrosis using acoustic radiation force-based shear stiffness in patients with nonalcoholic fatty liver disease

BACKGROUND & AIMS Nonalcoholic fatty liver disease (NAFLD), the most common form of chronic liver disease in developed countries, may progress to nonalcoholic steatohepatitis (NASH) in a minority of people. Those with NASH are at increased risk for cirrhosis and hepatocellular carcinoma. The potential risk and economic burden of utilizing liver biopsy to stage NAFLD in an overwhelmingly large at-risk population are enormous; thus, the discovery of sensitive, inexpensive, and reliable noninvasive diagnostic modalities is essential for population-based screening. METHODS Acoustic Radiation Force Impulse (ARFI) shear wave imaging, a noninvasive method of assessing tissue stiffness, was used to evaluate liver fibrosis in 172 patients diagnosed with NAFLD. Liver shear stiffness measures in three different imaging locations were reconstructed and compared to the histologic features of NAFLD and AST-to-platelet ratio indices (APRI). RESULTS Reconstructed shear stiffnesses were not associated with ballooned hepatocytes (p=0.11), inflammation (p=0.69), nor imaging location (p=0.11). Using a predictive shear stiffness threshold of 4.24kPa, shear stiffness distinguished low (fibrosis stage 0-2) from high (fibrosis stage 3-4) fibrosis stages with a sensitivity of 90% and a specificity of 90% (AUC of 0.90). Shear stiffness had a mild correlation with APRI (R(2)=0.22). BMI>40kg/m(2) was not a limiting factor for ARFI imaging, and no correlation was noted between BMI and shear stiffness (R(2)=0.05). CONCLUSIONS ARFI imaging is a promising imaging modality for assessing the presence or absence of advanced fibrosis in patients with obesity-related liver disease.

Hedgehog-Mediated Epithelial-to-Mesenchymal Transition and Fibrogenic Repair in Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Repair responses define the ultimate outcomes of liver disease. This study evaluated the hypothesis that fibrogenic repair in nonalcoholic fatty liver disease (NAFLD) is mediated by Hedgehog (Hh) pathway activation and consequent induction of epithelial-to-mesenchymal transitions (EMT) in ductular-type progenitors. METHODS Immature ductular cells were exposed to Sonic hedgehog (Shh) in the presence or absence of the Hh inhibitor cyclopamine to determine whether Hh-pathway activation directly modulates EMT in liver progenitors. Potential biologic correlates of progenitor cell EMT were assessed using mice fed methionine-choline-deficient + ethionine (MCDE) diets with or without cyclopamine. The effects of increased Hh signaling on EMT and fibrogenic repair during diet-induced NAFLD were also compared in wild-type (WT) and Patched haplo-insufficient (Ptc(+/-)) mice. Finally, evidence of Hh-pathway activation and EMT was examined in liver sections from patients with NAFLD. RESULTS In cultured progenitors, Shh repressed expression of epithelial genes and EMT inhibitors but induced genes that are expressed by myofibroblasts. Cyclopamine reversed these effects. In mouse NAFLD models, Hh-pathway activation, EMT, expansion of myofibroblastic populations, and liver fibrosis occurred. Cyclopamine inhibited Hh-pathway activation and induction of EMT. Ptc(+/-) mice, which have an overactive Hh pathway, exhibited sustained overinduction of Hh target genes and more EMT, myofibroblast accumulation, and fibrosis than WT mice. Numbers of Shh-producing cells and Hh-responsive ductular cells that expressed EMT markers increased in parallel with liver fibrosis in patients with NAFLD. CONCLUSIONS Hh-mediated EMT in ductular cells contributes to the pathogenesis of cirrhosis in NAFLD.

Betaine for nonalcoholic fatty liver disease: Results of a randomized placebo‐controlled trial

Based on animal studies and pilot studies in humans, betaine, a methyl donor for the remethylation of homocysteine, may be a therapeutic agent for nonalcoholic steatohepatitis (NASH). We evaluated the safety and efficacy of betaine for patients with NASH and whether betaine positively modified factors postulated to be “second hits” and underlying mechanisms of NASH. We conducted a randomized placebo‐control study of 55 patients with biopsy‐proven NASH who received either oral betaine (20 g daily) or placebo for 12 months. Pre‐ and posttreatment variables were analyzed using the paired t test or Wilcoxon rank test. Treatment groups were comparable at baseline. Of the 35 patients (17 betaine, 18 placebo) who completed the study, 34 patients (16 betaine, 18 placebo) underwent posttreatment liver biopsy. Patients randomized to betaine had a decrease in steatosis grade. No intra‐ or intergroup differences or changes in nonalcoholic fatty liver disease activity score or fibrosis stage were noted. Elevations of insulin, glucose, and proinflammatory cytokines and the reduced antioxidant status noted in NASH patients did not improve with betaine therapy. The antiinflammatory agent adiponectin was significantly reduced in both groups and did not change with therapy. Lastly, S‐adenosylhomocysteine was approximately twice normal and was not reduced by betaine therapy. Conclusion: Compared to placebo, betaine improved hepatic steatosis and may protect against worsening steatosis. High‐dose betaine supplementation failed to reduce S‐adenosylhomocysteine and did not positively affect any of the second hit mechanisms postulated to contribute to NASH that we studied. Although betaine has been proven effective in treating hepatic steatosis in several animal models, translating novel therapeutic options noted in animal studies to humans with NASH will prove challenging. (HEPATOLOGY 2009.)

Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation.

Cyclosporine is an immunosuppressive agent widely used in the management of liver transplant recipients. Cyclosporine has been shown to have antiviral activities against HIV, herpes simplex, and vaccinia viruses. The aim of this study was to determine the effect of Cyclosporine in viral clearance in the liver transplant recipients during therapy with combination of interferon and ribavirin, and to determine the anti‐viral potential of Cyclosporine in vitro. Immunosuppression consisted of either Cyclosporine or Tacrolimus‐based therapy. Both groups received therapy with interferon and ribavirin for 48 weeks when evidence of progressive histologic disease was determined. We found that subjects on Cyclosporine‐based immunosuppression (n = 56) had a higher sustained virological response of 46% compared to 27% in the patients on Tacrolimus‐based therapy (n=59, P = 0.03). In vitro studies were performed to evaluate the antiviral effect of Cyclosporine in the replicon system. These studies showed that Cyclosporine inhibits hepatitis C viral replication in a dose‐dependent manner. Combination of Cyclosporine with interferon showed additive effect, and its function is independent of interferon signaling pathways. In conclusion, Cyclosporine may offer an advantage to Tacrolimus in those patients undergoing interferon‐based therapy and should be studied in a prospective randomized trial. Liver Transpl 12:51–57, 2006.

Relationship Between Methylome and Transcriptome in Patients With Nonalcoholic Fatty Liver Disease

BACKGROUND & AIMS Cirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting noninvasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression. METHODS We collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0-1 (mild) and F3-4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome. RESULTS Clinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P < .05). Methylation at fibroblast growth factor receptor 2, methionine adenosyl methyltransferase 1A, and caspase 1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, and genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and underexpressed. CONCLUSIONS Functionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.

Sustained viral response to interferon and ribavirin in liver transplant recipients with recurrent hepatitis C

Recurrent hepatitis C infection is an important cause of progressive fibrosis, cirrhosis, and graft loss following orthotopic liver transplantation. Treatment for posttransplant recurrence of hepatitis C with interferon‐based therapy is difficult but results in loss of detectable virus in up to 30% of patients. However, the durability of viral clearance and the associated histologic response in this setting is unknown. The aim of this study was to determine whether viral loss in response to antiviral therapy is durable and associated with improvement in liver histology. All liver transplant recipients who received interferon‐based treatment for recurrent hepatitis C virus (HCV) at the University of Florida from 1991 to 2002 were included in this study. Patients who lost detectable HCV after treatment with interferon alone or in combination with ribavirin were followed to assess the durability of viral response and its impact on liver histology. One hundred nineteen transplant recipients were treated with interferon or combination therapy. Twenty‐nine (20 men, 9 women; mean age, 54 yrs [range, 42–74 yrs]) lost detectable HCV RNA and remained virus negative for at least 6 months after discontinuing therapy (sustained viral response[SVR]). The mean follow‐up after discontinuing therapy was 24.7 months (range, 6–70 mos). Our study cohort included one patient with SVR following interferon monotherapy and 28 patients with SVR following combination therapy with interferon plus ribavirin. All patients remained HCV RNA negative (assessed by polymerase chain reaction or branched‐DNA assay) during follow‐up of up to 5 years. Liver histology assessed 2 years after treatment showed less inflammation compared with before treatment in 50% and showed no change in 38%. By 3 to 5 years post‐treatment (n = 15 recipients), inflammation was reduced in 60% and remained unchanged in 33%. Fibrosis stage at 2 years improved by ≥ 1 stage in 27 %, remained unchanged in 38 %, and worsened in 35% despite viral clearance. At 3 to 5 years, the fibrosis stage had improved in 67%, remained unchanged in 13%, and worsened in 20%. Both grade of inflammation and fibrosis stage improved by 3 to 5 years posttreatment compared with baseline histology (p < 0.05). In conclusion, loss of HCV after treatment of recurrent chronic hepatitis C with interferon and ribavirin is durable, and the durability of the SVR is associated with improvement in hepatic inflammation and regression of fibrosis. (Liver Transpl 2004;10:199–207.)