Eleonora Scorletti

Effects of purified eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: Results from the WELCOME* study

There is no licensed treatment for nonalcoholic fatty liver disease (NAFLD), a condition that increases risk of chronic liver disease, type 2 diabetes, and cardiovascular disease. We tested whether 15‐18 months of treatment with docosahexaenoic acid (DHA) plus eicosapentaenoic acid (EPA; Omacor/Lovaza, 4 g/day) decreased liver fat and improved two histologically validated liver fibrosis biomarker scores (primary outcomes). Patients with NAFLD were randomized in a double‐blind, placebo‐controlled trial (DHA+EPA, n = 51; placebo, n = 52). We quantified liver fat percentage by magnetic resonance spectroscopy in three liver zones. We measured liver fibrosis using two validated scores. We tested adherence to the intervention (Omacor group) and contamination (with DHA and EPA; placebo group) by measuring erythrocyte percentage DHA and EPA enrichment (gas chromatography). We undertook multivariable linear regression to test effects of (1) DHA+EPA treatment (intention‐to‐treat analyses) and (2) erythrocyte DHA and EPA enrichment (secondary analysis). Median (interquartile range) baseline and end‐of‐study liver fat percentage were 21.7 (19.3) and 19.7 (18.0) (placebo) and 23.0 (36.2) and 16.3 (22.0) (DHA+EPA). In the fully adjusted regression model, there was a trend toward improvement in liver fat percentage with DHA+EPA treatment (β = −3.64; 95% confidence interval [CI]: −8.0, 0.8; P = 0.1), but there was evidence of contamination in the placebo group and variable adherence to the intervention in the Omacor group. Further regression analysis showed that DHA enrichment was independently associated with a decrease in liver fat percentage (for each 1% enrichment: β = −1.70; 95% CI: −2.9, −0.5; P = 0.007). No improvement in fibrosis scores occurred. Conclusion: Erythrocyte DHA enrichment with DHA+EPA treatment is linearly associated with decreased liver fat percentage. Substantial decreases in liver fat percentage can be achieved with high‐percentage erythrocyte DHA enrichment in NAFLD. (Hepatology 2014;60:1211–1221)

Omega-3 Fatty Acids, Hepatic Lipid Metabolism, and Nonalcoholic Fatty Liver Disease

Long-chain omega-3 fatty acids belong to a family of polyunsaturated fatty acids that are known to have important beneficial effects on metabolism and inflammation. Such effects may confer a benefit in specific chronic noncommunicable diseases that are becoming very prevalent in Westernized societies [e.g., nonalcoholic fatty liver disease (NAFLD)]. Typically, with a Westernized diet, long-chain omega-6 fatty acid consumption is markedly greater than omega-3 fatty acid consumption. The potential consequences of an alteration in the ratio of omega-6 to omega-3 fatty acid consumption are increased production of proinflammatory arachidonic acid-derived eicosanoids and impaired regulation of hepatic and adipose function, predisposing to NAFLD. NAFLD represents a spectrum of liver fat-related conditions that originates with ectopic fat accumulation in liver (hepatic steatosis) and progresses, with the development of hepatic inflammation and fibrosis, to nonalcoholic steatohepatitis (NASH). If the adipose tissue is inflamed with widespread macrophage infiltration, the production of adipokines may act to exacerbate liver inflammation and NASH. Omega-3 fatty acid treatment may have beneficial effects in regulating hepatic lipid metabolism, adipose tissue function, and inflammation. Recent studies testing the effects of omega-3 fatty acids in NAFLD are showing promise and suggesting that these fatty acids may be useful in the treatment of NAFLD. To date, further research is needed in NAFLD to (a) establish the dose of long-chain omega-3 fatty acids as a treatment, (b) determine the duration of therapy, and (c) test whether there is benefit on the different component features of NAFLD (hepatic fat, inflammation, and fibrosis).

Non-alcoholic fatty liver disease and cardiovascular risk: metabolic aspects and novel treatments

Non-alcoholic fatty liver disease (NAFLD) is usually a silent disease that occurs in a very high proportion of people with features of the metabolic syndrome, including overweight, insulin resistance and type 2 diabetes. Because obesity and type 2 diabetes are now extremely common in Westernised societies, it is likely that the prevalence of NAFLD increases markedly in the future. Although previously it was thought that NAFLD was harmless, it is now recognised that NAFLD can be a progressive liver condition that increases risk of cirrhosis, end-stage liver disease and hepatocellular carcinoma. Additionally, liver fat accumulation causes insulin resistance and increases risk of type 2 diabetes. Increasing evidence now shows NAFLD is a risk factor for cardiovascular disease (CVD). The purpose of this review is to briefly discuss the pathogenesis of NAFLD, to describe the relationship between NAFLD and CVD and the mechanisms linking both conditions and to discuss some of the treatment options (including lifestyle, nutrition and drugs) that may influence both NAFLD and risk of CVD.

Treating liver fat and serum triglyceride levels in NAFLD, effects of PNPLA3 and TM6SF2 genotypes: Results from the WELCOME trial

BACKGROUND & AIMS Genetic variation in both patatin-like phospholipase domain-containing protein-3 (PNPLA3) (I148M) and the transmembrane 6 superfamily member 2 protein (TM6SF2) (E167K) influences severity of liver disease, and serum triglyceride concentrations in non-alcoholic fatty liver disease (NAFLD), but whether either genotype influences the responses to treatments is uncertain. METHODS One hundred three patients with NAFLD were randomised to omega-3 fatty acids (DHA+EPA) or placebo for 15-18months in a double blind placebo controlled trial. Erythrocyte enrichment with DHA and EPA was measured by gas chromatography. PNPLA3 and TM6SF2 genotypes were measured by PCR technologies. Multivariable linear regression and analysis of covariance were undertaken to test the effect of genotypes on omega-3 fatty acid enrichment, end of study liver fat percentage and serum triglyceride concentrations. All models were adjusted for baseline measurements of each respective outcome. RESULTS Fifty-five men and 40 women (Genotypes PNPLA3 I148M, 148I/I=41, 148I/M=43, 148M/M=11; TM6SF2 E167K 167E/E=78, 167E/K+167K/K=17 participants) (mean ± SD age, 51 ± 11 years) completed the trial. Adjusting for baseline measurement, measured covariates and confounders, PNPLA3 148M/M variant was independently associated with percentage of DHA enrichment (B coefficient -1.02 (95% CI -1.97, -0.07), p=0.036) but not percentage of EPA enrichment (B coefficient -0.31 (95% CI -1.38, 0.75), p=0.56). This genotype was also independently associated with end of study liver fat percentage (B coefficient 9.5 (95% CI 2.53, 16.39), p=0.008), but not end of study triglyceride concentration (B coefficient -0.11 (95% CI -0.64, 0.42), p=0.68). CONCLUSIONS PNPLA3 148M/M variant influences the changes in liver fat and DHA tissue enrichment during the trial but not the change in serum triglyceride concentration.

Design and rationale of the WELCOME trial: a randomised, placebo controlled study to test the efficacy of purified long chain omega-3 fatty treatment in non-alcoholic fatty liver disease

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) represents a range of liver conditions from simple fatty liver to progressive end stage liver disease requiring liver transplantation. NAFLD is common in the population and in certain sub groups (e.g. type 2 diabetes) up to 70% of patients may be affected. NAFLD is not only a cause of end stage liver disease and hepatocellular carcinoma, but is also an independent risk factor for type 2 diabetes and cardiovascular disease. Consequently, effective treatments for NAFLD are urgently needed. OBJECTIVES The WELCOME study is testing the hypothesis that treatment with high dose purified long chain omega-3 fatty acids will have a beneficial effect on a) liver fat percentage and b) two histologically validated algorithmically-derived biomarker scores for liver fibrosis. DESIGN In a randomised double blind placebo controlled trial, 103 participants with NAFLD were randomised to 15-18months treatment with either 4g/day purified long chain omega-3 fatty acids (Omacor) or 4g/day olive oil as placebo. Erythrocyte percentage DHA and EPA enrichment (a validated proxy for hepatic enrichment) was determined by gas chromatography. Liver fat percentage was measured in three discrete liver zones by magnetic resonance spectroscopy (MRS). We also measured body fat distribution, physical activity and a range of cardiometabolic risk factors. METHODS Recruitment started in January 2010 and ended in June 2011. We identified 178 potential participants, and randomised 103 participants who met the inclusion criteria. The WELCOME study was approved by the local ethics committee (REC: 08/H0502/165; www.clinicalTrials.gov registration number NCT00760513).

Serum uric acid concentrations and fructose consumption are independently associated with NASH in children and adolescents

BACKGROUND & AIMS Recent research has suggested that dietary fructose intake may increase serum uric acid (UA) concentrations. Both UA concentration and fructose consumption maybe also increase in NAFLD. It is not known whether dietary fructose consumption and UA concentration are independently associated with non-alcoholic steatohepatitis (NASH). Our aim was to investigate the factors associated with NASH in children and adolescents with proven NAFLD, and to test whether UA concentrations and fructose consumption are independently associated with NASH. METHODS Obese children with NAFLD were studied (n=271). NASH was diagnosed by a NAFLD activity score ⩾5 and the fatty liver inhibition of progression (FLIP) algorithm. Fructose consumption (g/day) was assessed by food frequency questionnaire, and UA (mg/dl) was measured in serum. Binary logistic regression with adjustment for covariates and potential confounders was undertaken to test factors independently associated with NASH. RESULTS NASH occurred in 37.6% of patients. Hyperuricaemia (UA ⩾5.9mg/dl) was present in 47% of patients with NASH compared with 29.7% of non-NASH patients (p=0.003). Both UA concentration (OR=2.488, 95% CI: 1.87-2.83, p=0.004) and fructose consumption (OR=1.612, 95% CI 1.25-1.86, p=0.001) were independently associated with NASH, after adjustment for multiple (and all) measured confounders. Fructose consumption was independently associated with hyperuricaemia (OR=2.021, 95% CI: 1.66-2.78, p=0.01). These data were confirmed using the FLIP algorithm. CONCLUSIONS Both dietary fructose consumption and serum UA concentrations are independently associated with NASH. Fructose consumption was the only factor independently associated with serum UA concentration. LAY SUMMARY Currently, it is not known whether dietary fructose consumption and uric acid (UA) concentration are linked with non-alcoholic steatohepatitis (NASH) in children and adolescents. Our aim was to test whether UA concentrations and fructose consumption are independently associated with NASH in children and adolescents with proven non-alcoholic fatty liver disease (NAFLD). We show that both dietary fructose consumption and serum UA concentrations are independently associated with NASH and fructose consumption was independently linked with high serum UA concentrations.

Improvement in non-alcoholic fatty liver disease severity is associated with a reduction in carotid intima-media thickness progression

BACKGROUND AND AIMS n-3 polyunsaturated fatty acid (PUFA) treatment may decrease liver fat in non-alcoholic fatty liver disease (NAFLD), but uncertainty exists whether this treatment also decreases cardiovascular disease (CVD) risk in NAFLD. We tested whether 15-18 months n-3 PUFA [docosahexaenoic acid (DHA) and eicosapentaenoic acid] (Omacor/Lovaza, 4 g/day) vs placebo decreased carotid intima-media thickness (CIMT) progression, a surrogate marker of CVD risk. We also evaluated if improvement in markers of NAFLD severity was associated with decreased CIMT progression over time. METHODS In a pre-specified sub-study of the WELCOME (Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD with OMacor thErapy) trial (NCT00760513), CIMT was measured using B-mode ultrasound while NAFLD severity was assessed by measuring liver fat percentage (magnetic resonance spectroscopy) and hepatic necro-inflammation (serum cytokeratin-18 (CK-18) concentration), at baseline and end of study. RESULTS 92 patients (age 51.5 ± 10.7 years, 57.6% men) completed the study. In the treatment group (n = 45), CIMT progressed by 0.012 mm (IQR 0.005-0.020 mm) compared to 0.015 mm (IQR 0.007-0.025 mm) in the placebo group (n = 47) (p = 0.17). Reduced CIMT progression in the entire cohort was independently associated with decreased liver fat (standardized β-coefficient 0.32, p = 0.005), reduced CK-18 levels (standardized β-coefficient 0.22, p = 0.04) and antihypertensive usage (standardized β-coefficient -0.31, p = 0.009) in multivariable regression analysis after adjusting for all potential confounders. Decreased weight (standardized β-coefficient 0.30, p < 0.001) and increased DHA tissue enrichment during the 18-month study (standardized β-coefficient -0.19, p = 0.027) were both independently associated with decreased liver fat, but not with CK-18. CONCLUSION Improvement in two markers of NAFLD severity is independently associated with reduced CIMT progression.

Nonalcoholic Fatty Liver Disease and Reduced Serum Vitamin D3 levels

Nonalcoholic fatty liver disease (NAFLD) and vitamin D3 deficiency are two highly prevalent pathologic conditions worldwide that share several cardiometabolic risk factors. In addition to its traditional calcium-related effects on the skeleton, vitamin D3 deficiency has now been recognized to exert nonskeletal adverse effects on several other organ systems. Accumulating epidemiological evidence suggests that low levels of serum 25-hydroxyvitamin D3 are associated with the presence and severity of NAFLD, independently of several potential confounders, including features of the metabolic syndrome. The molecular mechanisms of this association remain incompletely understood. A variety of biologically plausible mechanisms may mediate a hepato-protective role for the active metabolite of vitamin D3. 1α,25-dihydroxyvitamin D3 modulates the insulin signaling pathway/insulin resistance, suppresses fibroblast proliferation and collagen production, exerts anticoagulant and profibrinolytic effects, and modulates macrophage activity and inflammatory cytokine generation. Overall, the high prevalence of vitamin D3 deficiency and the plausible biological mechanisms linking this to NAFLD suggest that treatment of vitamin D3 deficiency to prevent and/or treat NAFLD is a promising field to explore. Large placebo-controlled randomized clinical trials are urgently needed to determine whether vitamin D3 supplementation could have any potential benefit in reducing the development and progression of NAFLD.

Omega-3 fatty acids: Mechanisms of benefit and therapeutic effects in pediatric and adult NAFLD

Abstract Non-alcoholic fatty liver disease (NAFLD) is currently considered the most common liver disease in industrialized countries, and it is estimated that it will become the most frequent indication for liver transplantation in the next decade. NAFLD may be associated with moderate (i.e. steatosis) to severe (i.e. steatohepatitis and fibrosis) liver damage and affects all age groups. Furthermore, subjects with NAFLD may be at a greater risk of other obesity-related complications later in life, and people with obesity and obesity-related complications (e.g. metabolic syndrome, type 2 diabetes and cardiovascular disease) are at increased risk of developing NAFLD. To date, there is no licensed treatment for NAFLD and therapy has been mainly centered on weight loss and increased physical activity. Unfortunately, it is often difficult for patients to adhere to the advised lifestyle changes. Therefore, based on the known pathogenesis of NAFLD, several clinical trials with different nutritional supplementation and prescribed drugs have been undertaken or are currently underway. Experimental evidence has emerged about the health benefits of omega-3 fatty acids, a group of polyunsaturated fatty acids that are important for a number of health-related functions. Omega-3 fatty acids are present in some foods (oils, nuts and seeds) that also contain omega-6 fatty acids, and the best sources of exclusively omega-3 fatty acids are oily fish, krill oil and algae. In this review, we provide a brief overview of the pathogenesis of NAFLD, and we also discuss the molecular and clinical evidence for the benefits of different omega-3 fatty acid preparations in NAFLD.

Docosahexaenoic acid enrichment in NAFLD is associated with improvements in hepatic metabolism and hepatic insulin sensitivity: a pilot study

Background/Objective:Treatment of subjects with non-alcoholic fatty liver disease (NAFLD) with omega-3 polyunsaturated fatty acids (FAs) suggests high levels of docosahexaenoic acid (DHA) tissue enrichment decrease liver fat content. We assessed whether changes in erythrocyte DHA enrichment (as a surrogate marker of changes in tissue enrichment) were associated with alterations in hepatic de novo lipogenesis (DNL), postprandial FA partitioning and hepatic and peripheral insulin sensitivity in a sub-study of the WELCOME trial (Wessex Evaluation of fatty Liver and Cardiovascular markers in NAFLD (non-alcoholic fatty liver disease) with OMacor thErapy).Subjects/Methods:Sixteen participants were randomised to 4 g/day EPA+DHA (n=8) or placebo (n=8) for 15–18 months and underwent pre- and post-intervention measurements. Fasting and postprandial hepatic FA metabolism was assessed using metabolic substrates labelled with stable-isotope tracers (2H2O and [U13C]palmitate). Insulin sensitivity was measured by a stepped hyperinsulinaemic-euglycaemic clamp using deuterated glucose. Participants were stratified according to change in DHA erythrocyte enrichment (< or ⩾2% post intervention).Results:Nine participants were stratified to DHA⩾2% (eight randomised to EPA+DHA and one to placebo) and seven to the DHA<2% group (all placebo). Compared with individuals with erythrocyte <2% change in DHA abundance, those with ⩾2% enrichment had significant improvements in hepatic insulin sensitivity, reduced fasting and postprandial plasma triglyceride concentrations, decreased fasting hepatic DNL, as well as greater appearance of 13C from dietary fat into plasma 3-hydroxybutyrate (all P<0.05).Conclusions:The findings from our pilot study indicate that individuals who achieved a change in erythrocyte DHA enrichment ⩾2% show favourable changes in hepatic FA metabolism and insulin sensitivity, which may contribute to decreasing hepatic fat content.