Brent A. Neuschwander-Tetri

Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions

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.

Pioglitazone, Vitamin E, or Placebo for Nonalcoholic Steatohepatitis

BACKGROUND Nonalcoholic steatohepatitis is a common liver disease that can progress to cirrhosis. Currently, there is no established treatment for this disease. METHODS We randomly assigned 247 adults with nonalcoholic steatohepatitis and without diabetes to receive pioglitazone at a dose of 30 mg daily (80 subjects), vitamin E at a dose of 800 IU daily (84 subjects), or placebo (83 subjects), for 96 weeks. The primary outcome was an improvement in histologic features of nonalcoholic steatohepatitis, as assessed with the use of a composite of standardized scores for steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis. Given the two planned primary comparisons, P values of less than 0.025 were considered to indicate statistical significance. RESULTS Vitamin E therapy, as compared with placebo, was associated with a significantly higher rate of improvement in nonalcoholic steatohepatitis (43% vs. 19%, P=0.001), but the difference in the rate of improvement with pioglitazone as compared with placebo was not significant (34% and 19%, respectively; P=0.04). Serum alanine and aspartate aminotransferase levels were reduced with vitamin E and with pioglitazone, as compared with placebo (P<0.001 for both comparisons), and both agents were associated with reductions in hepatic steatosis (P=0.005 for vitamin E and P<0.001 for pioglitazone) and lobular inflammation (P=0.02 for vitamin E and P=0.004 for pioglitazone) but not with improvement in fibrosis scores (P=0.24 for vitamin E and P=0.12 for pioglitazone). Subjects who received pioglitazone gained more weight than did those who received vitamin E or placebo; the rates of other side effects were similar among the three groups. CONCLUSIONS Vitamin E was superior to placebo for the treatment of nonalcoholic steatohepatitis in adults without diabetes. There was no benefit of pioglitazone over placebo for the primary outcome; however, significant benefits of pioglitazone were observed for some of the secondary outcomes. (ClinicalTrials.gov number, NCT00063622.)

Nonalcoholic steatohepatitis: an expanded clinical entity.

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 virus-negative. 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.

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.

Humanized xenobiotic response in mice expressing nuclear receptor SXR

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-16α-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.

Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites.

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.

Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings

The diagnosis of nonalcoholic steatohepatitis (NASH) is defined by the presence and pattern of specific histological abnormalities on liver biopsy. A separate system of scoring the features of nonalcoholic fatty liver disease (NAFLD) called the NAFLD Activity Score (NAS) was developed as a tool to measure changes in NAFLD during therapeutic trials. However, some studies have used threshold values of the NAS, specifically NAS ≥5, as a surrogate for the histologic diagnosis of NASH. To evaluate whether this unintended use of the NAS is valid, biopsy and clinical data from the 976 adults in NASH Clinical Research Network (CRN) studies were reviewed. Biopsies were evaluated centrally by the NASH CRN Pathology Committee. Definite steatohepatitis (SH) was diagnosed in 58.1%, borderline SH in 19.5% and “not SH” in 22%. The NAS was ≥5 in 50% and ≤4 in 49%; in this cohort only 75% of biopsies with definite SH had an NAS ≥5, whereas 28% of borderline SH and 7% of “not SH” biopsies had NAS ≥5. Of biopsies with an NAS ≥5, 86% had SH and 3% “not SH”. NAS ≤4 did not indicate benign histology; 29% had SH and only 42% had “not SH.” Higher values of the NAS were associated with higher levels of alanine aminotransferase and aspartate aminotransferase, whereas the diagnosis of SH was associated with features of the metabolic syndrome. Conclusion: The diagnosis of definite SH or the absence of SH based on evaluation of patterns as well as individual lesions on liver biopsies does not always correlate with threshold values of the semiquantitative NAS. Clinical trials and observational studies should take these different performance characteristics into account. (HEPATOLOGY 2011)

Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease

Background: Clinical predictors of advanced non-alcoholic liver disease (NAFLD) are needed to guide diagnostic evaluation and treatment. Methods: 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. Results: The cohort was 51% female and had a median body mass index (BMI) of 33 kg/m2; 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, BMI ⩾28 kg/m2, age >50 years, and aspartate transaminase/alanine aminotransferase (AST/ALT) ratio ⩾0.8, a quantitative assessment check index (QUICKI) score <0.294 (equivalent to homeostatasis model assessment (HOMA) >6.2) and the presence of diabetes mellitus (DM) were individually associated by univariate analysis with odds ratios (ORs) of ⩾2.4 for advanced fibrosis. Based on the results of forced entry logistic regression analysis, three variables were combined in a weighted sum (BMI ⩾28 = 1 point, AAR of ⩾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%. Conclusions: 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.

Orlistat for overweight subjects with nonalcoholic steatohepatitis: A randomized, prospective trial

The aim of this study was to determine if orlistat, an inhibitor of fat absorption, combined with caloric restriction in overweight subjects with nonalcoholic steatohepatitis results in weight loss and improved liver histology. Fifty overweight subjects (body mass index = ≥27) with biopsy proven nonalcoholic steatohepatitis were randomized to receive a 1,400 Kcal/day diet plus vitamin E (800 IU) daily with or without orlistat (120 mg three times a day) for 36 weeks. Liver biopsies were repeated at week 36. Twenty‐three subjects in the orlistat/diet/vitamin E group and 18 in the diet/vitamin E group completed the study. The mean age was 47 ± 9.0 (standard deviation) years and mean body mass index was 36.4 ± 6.3 kg/m2. Four subjects were diabetic. The orlistat group lost a mean of 8.3% body weight compared to 6.0% in the diet plus vitamin E group (not significant). Both groups also had similarly improved serum aminotransferases, hepatic steatosis, necroinflammation, ballooning, and nonalcoholic fatty liver disease activity scores. Stratified according to weight loss instead of treatment group, a loss of ≥5% body weight (n = 24) compared to <5% body weight (n = 17) correlated with improvement in insulin sensitivity (P = 0.001) and steatosis (P = 0.015). Comparing subjects who lost ≥9% of body weight (n = 16), to those that did not (n = 25), improved insulin sensitivity (P < 0.001), adiponectin (P = 0.03), steatosis (P = 0.005), ballooning (P = 0.04), inflammation (P = 0.045), and nonalcoholic fatty liver disease activity score (P = 0.009) were seen. Increases in adiponectin strongly correlated with improved ballooning and nonalcoholic fatty liver disease activity score (P = 0.03). Orlistat did not enhance weight loss or improve liver enzymes, measures of insulin resistance, and histopathology. However, subjects who lost ≥5% of body weight over 9 months improved insulin resistance and steatosis, and those subjects who lost ≥9% also achieved improved hepatic histologic changes. (HEPATOLOGY > 2009;49:80‐86.)

Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent

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-alpha 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.