Helena Cortez-Pinto

A position statement on NAFLD/NASH based on the EASL 2009 special conference

Universite Pierre et Marie Curie Paris VI, Assistance Publique Hopitaux de Paris, INSERM UMRS 893, France; Centro Studi Fegato, Gastroenterologia, Distretto di Carpi, Azienda USL di Modena, Italy; Departamento de Gastrenterologia, Unidade de Nutricao e Metabolismo, Hospital Universitario de Santa Maria, Instituto de Medicina Molecular, Lisbon, Portugal; Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom; Alma Mater Studiorum, Unit of Metabolic Diseases and Clinical Dietetics, University of Bologna, Italy

Hepatocyte apoptosis, expression of death receptors, and activation of NF-κB in the liver of nonalcoholic and alcoholic steatohepatitis patients

OBJECTIVES:The increasing incidence of nonalcoholic (NASH) and alcoholic steatohepatitis (ASH), associated with lack of effective treatment, has prompted intensive studies on disease pathogenesis. Apoptosis is recognized as common in liver injury and may also contribute to tissue inflammation, fibrogenesis, and development of cirrhosis. In this study, we identified mechanisms of apoptosis induction in human steatohepatitis, and evaluated potential associations between apoptosis, liver pathology, and clinical presentation in NASH and ASH.METHODS:Hepatocyte apoptosis was evaluated by the TUNEL assay in 20 patients with NASH (all ambulatory), 40 with ASH (20 ambulatory, 20 hospitalized), and 20 controls. Liver biopsies were also graded for inflammation and fibrosis. Immunohistochemistry was performed for death receptors (Fas and TNF-R1), activated caspase-3, NF-κB p65, antiapoptotic Bcl-2 protein, and uncoupling protein 2 (UCP-2).RESULTS:TUNEL-positive hepatocytes were markedly increased in NASH (p < 0.05) and ASH (p < 0.01). Similar results were obtained for activated caspase-3, confirming the occurrence of apoptosis. The Fas receptor was upregulated in ASH, especially in hospitalized patients (p < 0.01), whereas TNF-R1 was expressed both in NASH and ASH (p < 0.01). In addition, patients with ASH showed a remarkable expression of active NF-κB, as compared to NASH and controls (p < 0.01). Degrees of inflammation and fibrosis correlated with NF-κB p65 expression, which in turn coincided with apoptosis albeit Bcl-2 and UCP-2 expression.CONCLUSIONS:Liver injury in NASH and ASH is associated with increased hepatocyte apoptosis mediated by death receptors. Further, apoptosis correlates with active NF-κB expression, and disease severity. This potential mechanistic link might provide multiple interesting targets for therapeutic intervention.

Non-alcoholic fatty liver: another feature of the metabolicsyndrome?

Abstract Background and aims: Hepatic steatosis and nonalcoholic steatohepatitis (NASH) have been associated with obesity, non insulin-dependent diabetes mellitus and hyperlipidemia. The present study was designed in order to evaluate whether patients with steatosis/NASH presented common features with the metabolic syndrome. Methods: In 30 patients with nonalcoholic fatty liver the prevalence of hypertension and diabetes; the glucose/insulin profile, lipid profile, and serum leptin were evaluated and correlated with body composition and energy expenditure, assessed by bioimpedance spectroscopy and indirect calorimetry, respectively. Results were compared with a group of eight controls. Results: Obesity was present in 80% of patients, hypertension in 50% and non insulin dependent diabetes in 33%. Glucose metabolism was altered in 69%, with elevated insulin in 14 patients. Serum leptin, higher in women, was increased in patients: 33.9 ± 38.9 vs 9.6 ± 6.9 ng/ml, P Conclusions: There is a strong association between nonalcoholic fatty liver and features of the metabolicsyndrome, suggesting a simultaneous insulin resistance and decreased sensitivity to leptin.

miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease

BACKGROUND & AIMS Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis (NASH). However, disease pathogenesis remains largely unknown. microRNA (miRNA or miR) expression has recently been reported to be altered in human NASH, and modulated by ursodeoxycholic acid (UDCA) in the rat liver. Here, we aimed at evaluating the miR-34a/Sirtuin 1(SIRT1)/p53 pro-apoptotic pathway in human NAFLD, and to elucidate its function and modulation by UDCA in the rat liver and primary rat hepatocytes. METHODS Liver biopsies were obtained from NAFLD morbid obese patients undergoing bariatric surgery. Rat livers were collected from animals fed a 0.4% UDCA diets. Primary rat hepatocytes were incubated with bile acids or free fatty acids (FFAs) and transfected with a specific miRNA-34a precursor and/or with a p53 overexpression plasmid. p53 transcriptional activity was assessed by ELISA and target reporter constructs. RESULTS miR-34a, apoptosis and acetylated p53 increased with disease severity, while SIRT1 diminished in the NAFLD liver. UDCA inhibited the miR-34a/SIRT1/p53 pathway in the rat liver in vivo and in primary rat hepatocytes. miR-34a overexpression confirmed its targeting by UDCA, which prevented miR-34a-dependent repression of SIRT1, p53 acetylation, and apoptosis. Augmented apoptosis by FFAs in miR-34a overexpressing cells was also inhibited by UDCA. Finally, p53 overexpression activated miR-34a/SIRT1/p53, which in turn was inhibited by UDCA, via decreased p53 transcriptional activity. CONCLUSIONS Our results support a link between liver cell apoptosis and miR-34a/SIRT1/p53 signaling, specifically modulated by UDCA, and NAFLD severity. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide new tools for therapeutic intervention.

Blood oxidative stress markers in non-alcoholic steatohepatitis and how it correlates with diet.

Objective. Non-alcoholic fatty liver disease is a common condition that can progress to endstage liver disease. The steatotic liver seems to be particularly susceptible to oxidative stress damage. The aim of this study was to evaluate the redox state in patients with non-alcoholic steatohepatitis (NASH) and its correlation with dietary intake. Material and methods. Plasma concentrations of 4-hydroxynonenal (4-HNE), 8-hydroxydeoxyguanosine (8-OHdG), reduced and oxidized glutathione (GSH and GSSG), vitamins A and E, total antioxidant status (TAS), glutathione peroxidase (GSH-Px) and reductase (GSH-Red) erythrocyte activities were compared between 43 NASH patients and 33 healthy controls. 4-HNE, GSH-Px, GSH-Red and TAS were evaluated by spectrophotometry, 8-OHdG by ELISA assay, GSH and GSSG by fluorimetric assay and vitamins A and E by high performance liquid chromatography. Dietary habits were also evaluated in these patients. Results. GSH levels (21.1±18.3 versus 33.1±22.2 µM, p=0.01) and GSH/GSSG ratio (0.9±0.7 versus 1.5±0.8, p=0.01) were lower and TAS (832±146 versus 630±140 µM, p<0.001) and vitamin E (47.1±14.9 versus 34.5±7.3 µM, p<0.001) were higher in NASH patients, although there was no difference in GSH-Px and GSH-Red activities, 8-OHdG and 4-HNE levels between groups. After adjusting for total energy consumption, a negative correlation was found with total and saturated fat intake and GSH/GSSG ratio, and a positive correlation with carbohydrates, fiber, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), specifically N-3 PUFA, and vitamins E, C, selenium and folate. Conclusions. Our data suggest an impaired glutathione metabolism towards an oxidant status in NASH patients, correlating with a higher intake of saturated fat and a lower intake of carbohydrates. Plasmatic concentrations of oxidative stress cellular markers did not translate to hepatic oxidative damage.

Nonalcoholic steatohepatitis: A long-term follow-up study. Comparison with alcoholic hepatitis in ambulatory and hospitalized patients

A previous publication analyzed the clinicopathological characteristics of 105 patients with steatohepatitis: 32 nonalcoholic, 21 ambulatory alcoholics, and 52 hospitalized alcoholics; we now report an up to 12-year follow-up (mean 5.9 ± 4.7). Between 1988 and 1993, all patients with a histological diagnosis of steatohepatitis were included; necrosis, inflammation, Mallory bodies, and fibrosis were graded. Complete follow-up data were obtained in 78%. Survival curves were similar between nonalcoholic and ambulatory alcoholics; they were, however, better in nonalcoholic than hospitalized alcoholics (P < 0.0001), and in ambulatory relative to hospitalized (P = 0.0001) alcoholics. Nonalcoholics had a better prognosis than the combined alcoholic groups (P = 0.001). Patients with moderate to severe Mallory bodies and severe fibrosis had a significantly worse survival (P < 0.01), whereas severity of hepatocellular damage and neutrophil or mononuclear infiltration had no significant impact. In conclusion, alcoholic patients as a whole had a worse prognosis, yet the ambulatory subgroup had a prognosis similar to nonalcoholic patients.

Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis

Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3(-/-)) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.

How good is controlled attenuation parameter and fatty liver index for assessing liver steatosis in general population: correlation with ultrasound

Liver steatosis measurement by controlled attenuation parameter (CAP) is a non‐invasive method for diagnosing steatosis, based on transient elastography. Its usefulness as screening procedure for hepatic steatosis in general population has not been previously evaluated. The aim of this study was to evaluate the diagnostic accuracy of CAP and fatty liver index (FLI) for detection and quantification of steatosis in general population.

Treatment and prognostic factors in patients with hepatocellular carcinoma

Abstract: Introduction: Hepatocellular carcinoma is a leading cause of death from cancer worldwide. Survival of patients depends on tumor extension and liver function, but yet there is no consensual prognostic model.

Cell death and nonalcoholic steatohepatitis: where is ballooning relevant?

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease in the Western world. Progression to more aggressive forms of liver injury, such as nonalcoholic steatohepatitis (NASH) and cirrhosis, occurs in less than a third of affected subjects. Human data and both in vivo and in vitro models demonstrate that cell death, particularly apoptosis, is increased in NAFLD and NASH patients, suggesting that it is crucial in disease progression. Indeed, fatty acids – more specifically, saturated fatty acids – strongly induce hepatocyte apoptosis. In addition, hepatic steatosis renders hepatocytes more susceptible to apoptotic injury. Ballooned hepatocytes and Mallory–Denk bodies are important hallmarks of NASH and correlate with disease progression. There are complex correlations between ballooning, Mallory–Denk bodies and apoptosis through keratin metabolism and depletion, as well as through the endoplasmic reticulum stress response. Whether apoptosis may promote hepatocellular ballooning, or vice versa, will be discussed in this article.