Exercise Reduces Liver Lipids and Visceral Adiposity in Patients With Nonalcoholic Steatohepatitis in a Randomized Controlled Trial

Accepted Manuscript Exercise Reduces Liver Lipids and Visceral Adiposity in Patients with Non-alcoholic Steatohepatitis in a Randomized Controlled Trial David Houghton, Christian Thoma, Kate Hallsworth, Sophie Cassidy, Timothy Hardy, Alastair D. Burt, Dina Tiniakos, Kieren G. Hollingsworth, Roy Taylor, Christopher P. Day, Stuart McPherson, Quentin M. Anstee, Michael I. Trenell PII: DOI: Reference:

S1542-3565(16)30512-2 10.1016/j.cgh.2016.07.031 YJCGH 54854

To appear in: Clinical Gastroenterology and Hepatology Accepted Date: 29 July 2016 Please cite this article as: Houghton D, Thoma C, Hallsworth K, Cassidy S, Hardy T, Burt AD, Tiniakos D, Hollingsworth KG, Taylor R, Day CP, McPherson S, Anstee QM, Trenell MI, Exercise Reduces Liver Lipids and Visceral Adiposity in Patients with Non-alcoholic Steatohepatitis in a Randomized Controlled Trial, Clinical Gastroenterology and Hepatology (2016), doi: 10.1016/j.cgh.2016.07.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Exercise Reduces Liver Lipids and Visceral Adiposity in Patients with Non-alcoholic

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Steatohepatitis in a Randomized Controlled Trial

3 David Houghton1, Christian Thoma1, Kate Hallsworth1, Sophie Cassidy1, Timothy

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Hardy1,2, Alastair D Burt3, Dina Tiniakos1,2, Kieren G Hollingsworth1, Roy Taylor1,

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Christopher P Day1,2, Stuart McPherson 1,2 Quentin M Anstee1,2, Michael I Trenell1

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Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK

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Liver Unit, Newcastle Upon Tyne Hospitals NHS Trust, Freeman Hospital, Newcastle

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Upon Tyne, UK.

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North Terrace, Adelaide, SA 5005, Australia

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Faculty of Health Sciences, The University of Adelaide, Level 2, Barr Smith South,

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Corresponding Author:

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Professor Michael Trenell, 4th Floor William Leech Building, Newcastle

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University, Newcastle upon Tyne, NE2 4HH, UK. Telephone: + 44 191 2086935

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Fax: + 44 191 2085685 Email: michael.trenell[at]ncl.ac.uk

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Clinical Trial Registration Number: ISRCTN16070927

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http://www.isrctn.com/ISRCTN16070927

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Word Count: 3568

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Short Title: An exercise study in NASH patients: a RCT

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Number of Figures and Tables: 2 figures; 4 tables

24 List of abbreviations:

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ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the

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curve; BMI, body mass index; CK-18, cytoketatin-18; ELF, enhanced liver fibrosis;

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fsOGTT, frequently sampled oral glucose tolerance test; HbA1c, glycated haemoglobin;

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hsCRP, high sensitivity C-reactive protein; IL-6, interleukin 6; HOMA-IR, homeostatic

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model assessment of insulin resistance; NAFLD, non-alcoholic fatty liver disease;

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NASH; non-alcoholic steatohepatitis; HTGC, hepatic triglyceride content; ¹H-MRS,

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proton magnetic resonance spectroscopy; SAT, subcutaneous adipose tissue; TNF-α,

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tumor necrosis factor alpha; T2DM, Type 2 diabetes mellitus; VAT, visceral adipose

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tissue.

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Conflict of Interest: None of the authors have any conflicts of interest.

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Financial Support:

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This research has received funding from the European Union Seventh Framework

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Programme (FP7/2007-2013) under grant agreement n° Health-F2-2009-241762, for the

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FLIP project; The Medical Research Council; Diabetes UK; The Newcastle Centre for

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Ageing and Vitality; The UK National Institute for Health Research Biomedical Research

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Centre on Ageing & Age Related Diseases; MIT was supported by a Senior Fellowship

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from the National Institute for Health Research.

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Author Contributions:

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DH: Study concept and design, study execution, data collection, statistical analysis,

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manuscript preparation, and approval of final draft submitted.

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ACCEPTED MANUSCRIPT CT: Study concept and design, study execution, data collection, statistical analysis,

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manuscript preparation, and approval of final draft submitted.

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KH: Study concept and design, study execution, data collection, statistical analysis,

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manuscript preparation, and approval of final draft submitted.

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SC: Study concept and design, critical revision of the paper and approval of final draft

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submitted.

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TH: Study concept and design, critical revision of the paper and approval of final draft

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submitted.

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ADB: Study concept and design, review of histology, critical revision of the paper and

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approval of the final draft submitted.

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DT: Review of histology, critical revision of the paper and approval of the final draft

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submitted.

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KGH: Study concept and design, review and analysis of magnetic resonance techniques,

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critical revision of the paper and approval of the final draft submitted.

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RT: Study concept and design, critical revision of the paper and approval of the final

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draft submitted.

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CPD: Study concept and design, critical revision of the paper and approval of the final

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draft submitted.

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SM: Study concept and design, critical revision of the paper and approval of final draft

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submitted.

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QMA: Study concept and design, critical revision of the paper and approval of final draft

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submitted.

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MIT: Study concept and design, statistical analysis, manuscript preparation and approval

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of the final draft submitted.

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ACCEPTED MANUSCRIPT Abstract

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Background & Aims: Pharmacologic treatments for non-alcoholic steatohepatitis

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(NASH) are limited. Lifestyle interventions are believed to be effective in reducing

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features of NASH, although the effect of regular exercise, independent of dietary change,

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is unclear. We performed a randomized controlled trial to study the effect of exercise on

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hepatic triglyceride content (HTGC) and biomarkers of fibrosis in patients with NASH.

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Methods: Twenty-four patients (mean age 52 ± 14 years; body mass index, 33 ± 6 kg/m–

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and biopsy-proven NASH received were randomly assigned to groups that exercised

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(n=12) or continued standard care (controls, n=12) for 12 weeks while maintaining their

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weight. The exercise (cycling and resistance training) was supervised at an accredited

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sports center and supervised by a certified exercise specialist and recorded 3 times per

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week, on non-consecutive days. We measured HTGC, body composition, circulating

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markers of inflammation, fibrosis, and glucose tolerance at baseline and at 12 weeks.

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) with sedentary lifestyles (less than 60 minutes of moderate–vigorous activity per week)

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Results: Compared with baseline, exercise significantly reduced HTGC (reduction of

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16%±24% vs an increase of 9%±15% for controls; P<.05), visceral fat (reduction of

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22±33 cm2 vs an increase of 14±48 cm2 for controls; P<.05), plasma triglycerides

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(reduction of 0.5±1.0 mmol/L vs an increase of 0.3±0.4 mmol/L for controls; P<.05), and

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gamma-glutamyltransferase (reduction of 10±28 U/L–1 vs a reduction of 17±38 U/L–1 for

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controls; P<.05). There were no effects of exercise on liver enzymes, metabolic

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parameters, circulatory markers of inflammation (levels of interleukin-6, tumor necrosis

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factor-α, or c-reactive protein) and fibrosis.

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ACCEPTED MANUSCRIPT Conclusion: In a randomized controlled trial, 12 weeks of exercise significantly reduced

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HTGC, visceral fat, and plasma triglycerides in patients with NASH, but did not affect

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circulating markers of inflammation or fibrosis. Exercise without weight loss therefore

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affects some but not all factors associated with NASH. Clinical care teams should

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consider exercise as part of a management strategy of NASH, but weight management

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strategies should be included. Larger and longer-term studies are required to determine

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the effects of exercise in patients with NASH. ISRCTN registry.com no:

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ISRCTN16070927.

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Clinical Trials Website and number: ISRCTNregistry.com, number

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ISRCTN16070927.

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Keywords: NAFLD; inflammation; therapy; body composition

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ACCEPTED MANUSCRIPT Introduction

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Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver conditions

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ranging from simple steatosis through non-alcoholic steatohepatitis (NASH), fibrosis and

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cirrhosis 1. Current evidence from a large single-centre serial biopsy study, supported by

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a recent meta-analysis, indicates that approximately 40% of NAFLD cases will exhibit

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progressive fibrosis during a median 6.6 year follow-up 2, 3. Although the accumulation of

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various lipids in the liver is the prerequisite for NAFLD, recent evidence suggests that

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inflammatory biomarkers could also play a role in the development of NASH 4.

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Currently there are no approved pharmacological therapies for managing NAFLD,

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although a number of promising agents are in trial 5. Lifestyle interventions,

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incorporating weight loss and increased physical activity/exercise, remain the cornerstone

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of NAFLD management 6-9, however, implementation remains difficult 10. Evidence

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supporting the effect of physical activity independent of weight loss is limited, impeding

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successful translation into clinical practice. Increasing physical activity through

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structured exercise has demonstrated a significant beneficial effect on HTGC 7, 11, 12. To

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date, only one study has assessed the effect of exercise in biopsy proven NASH and

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reported no change in HTGC or fibrosis 9. However, the study did not include a control

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group and used % hepatocytes effected to assess HTGC, limiting sensitivity.

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The primary objective of this randomised controlled trial was to determine the effects of

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exercise, without weight loss, on HTGC in adults with biopsy confirmed NASH. The

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secondary aims were to determine the effect of exercise on mediators of NASH;

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abdominal adiposity, glucose control, circulating markers of inflammation, and non-

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invasive markers of fibrosis.

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ACCEPTED MANUSCRIPT Materials and Methods:

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Thirty-one patients with histologically characterised NASH (age 59 ± 12years, BMI 35 ±

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5kg/m-2) were screened for study entry. Patients with evidence of other liver disease or a

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history of excessive alcohol consumption (alcohol intake >20g/day for women; >30g/day

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for men) were excluded. The study protocol was approved by the Sunderland Research

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Ethics Committee, UK and all patients gave written informed consent. Liver biopsies

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were histologically scored by two expert histopathologists (ADB and DT) according to

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the NASH Clinical Research Network criteria 13. NAFLD activity score (NAS) was

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graded between 0-8, and fibrosis was staged from 0 to 4 as previously performed 14

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(Table 2). Other exclusion criteria included: heart or kidney disease; implanted ferrous

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metal; pre-existing medical conditions preventing participation in the exercise

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programme; insulin sensitising treatment or dietary change over the preceding six

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months. Five patients were excluded during screening due to abnormal

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electrocardiographs and one withdrew due to claustrophobia during MRI scanning (see

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CONSORT diagram, Figure 1).

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Twenty six sedentary (<60 minutes of moderate-vigorous activity per week) patients

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were randomly assigned using a permuted blocks method to either exercise (n = 13) or

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standard care (n = 13) (Figure 1). Participant characteristics are summarised in Table 1.

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Following an initial screening visit, patients underwent a full medical history, physical

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examination and progressive exercise test to screen for any undiagnosed cardiac disease

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as previously described 12. HTGC, body composition, fasted blood samples, including

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inflammatory (IL-6, TNF-α and hsCRP (V-PLEX K15049D plate, Meso-Scale,

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Rockville, USA and Roche Diagnostics Ltd, Burgess Hill, UK, respectively) and fibrosis

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markers (CK-18 using M30-Apoptosense ELISA kit (PEVIVA, Bromma, Sweden) 15 and

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a 2h fsOGTT were measured at baseline and 12 weeks 12, 16, 17 (methods detailed in

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Online Supplement 1).

164 Study Intervention: Exercise was supervised by an accredited exercise specialist and

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recorded to ensure adherence, three times per week on non-consecutive days for 12

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weeks 18 19. The exercise programme consisted of aerobic (cycling) and resistance

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training and is detailed in online Supplement 1. All patients were instructed not to alter

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their diet and maintain current weight throughout the study.

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Statistics: Sample size was calculated based on change in HTGC from previous data in

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NAFLD 12; an 80% power of detecting a 10% relative difference between group change

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in liver HTGC with a SD of 9.0 and one-sided 0.05 significance required n=11 per group.

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We recruited 13 per sample to allow 2 drop outs per group. Normality was assessed using

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a Kolmogorov–Smirnov test and logarithmically transformed if not normally distributed.

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Between group differences were evaluated using an unpaired t-test and within group

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differences using a paired t-test (two way). Treatment x Time interactions were assessed

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using a two-way ANOVA. ANCOVA’s were employed to test for between group

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differences in outcome variables whilst controlling for baseline values . Bivariate

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correlations using Pearson rank correlations were conducted to investigate any

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associations between HTGC, body composition, triglycerides, glucose control,

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biomarkers of inflammation and NAFLD fibrosis marking systems. Statistical

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significance was set at p<0.05. Statistical analyses were performed using SPSS statistical

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analysis software (Version 19, IBM, USA). All authors had access to study data and had

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reviewed and approved the final manuscript.

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Twenty six patients were randomised with two withdrawing due to pre-existing knee and

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back problems (Figure 1). Twenty-four patients completed the study, with all subjects in

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the exercise group completing the 36-session exercise programme. Control and exercise

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groups were well matched (Table 1) with no significant differences in age (51 ± 16 vs. 54

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± 12years) or BMI (33 ± 5 vs. 33 ± 7kg/m-2). Baseline histological scores and fibrosis

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staging are presented in Table 2.

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BMI, body composition & HTGC

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During the study BMI, weight and subcutaneous fat remained constant in both groups

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(Tables 1 and 3). Visceral fat decreased by 12% in the exercise group and increased by

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7% in the control group (p<0.01; Figure 2 & Table 3). Exercise increased lean body mass

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by 4% vs. 0% in the control group (p<0.05), and reduced fat mass by 6% vs. 0% in the

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control group, although the latter was not statistically significant. Exercise produced a

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16% reduction in HTGC compared with an 8% increase in the control group (p<0.05;

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Figure 2 & Table 3).

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Blood lipids & liver enzymes

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There was a time by treatment interaction for triglycerides, with exercise eliciting a

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reduction of 23% vs. 13% increase in the control group (p<0.05). There was also a

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significant reduction of 13% in GGT following exercise (p<0.05). No other significant

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changes in blood biochemistry were observed in either group (Table 1).

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ACCEPTED MANUSCRIPT 212 Metabolic control

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The exercise group demonstrated a -17% vs. -7% reduction in fasting insulin, and

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decreased HOMA-IR -17% vs. +6%, although these did not reach statistical significance

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(Table 3). There were no differences in any other glucose control variables following the

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intervention in either group or between groups (Table 3). No time by treatment

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interaction for fasting blood glucose, fasting insulin, HbA1c, HOMA-IR and glucose

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AUC for fsOGTT were observed (Table 3).

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Circulatory inflammation & non-invasive fibrosis markers

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There was no significant time by treatment interaction for exercise on circulating

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inflammatory biomarkers, CK-18, hsCRP or the non-invasive markers of fibrosis

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(NAFLD Fibrosis score, AST/ALT ratio and ELF test) (Table 3).

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HTGC was positively associated with visceral fat at baseline and post intervention (r =

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0.49, p= 0.01 and r = 0.36, p= 0.04, respectively). There was also a positive correlation

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between the changes in HTGC and visceral fat following the intervention (r = 0.39, p=

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0.03). There were no other significant correlations between HTGC, body composition,

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glucose control, biomarkers of inflammation and fibrosis markers at baseline or post

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intervention.

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ACCEPTED MANUSCRIPT Discussion This is the first RCT to examine the effects of exercise, independent of weight loss, in adults with histologically confirmed NASH. The data shows that 12 weeks of exercise resulted in: 1) a 16% reduction in liver fat, 2) a 12% reduction in visceral fat, 3) a 23%

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reduction circulating triglycerides, and 4) a 4% increase in lean body mass. However, 12 weeks of exercise had no significant effect on glucose control, circulating markers of

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inflammation, liver enzymes or NAFLD activity score.

Lifestyle modification combining dietary change and exercise produces a robust

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reduction in HTGC and even reversal of inflammation with over 10% weight loss in people with NASH 6, 8, data on exercise without weight loss is lacking. We demonstrate that 12 weeks of exercise therapy, resulted in a 16% reduction in HTGC, independent of weight loss in people with NASH. The changes in HTGC reported here are in line with

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previous reports of exercise without weight loss in people with NAFLD 11, 12 and demonstrate the effectiveness of exercise in reducing HTGC in NASH. Although consistent with earlier reports in NAFLD, our observation contrasts with the only other

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study looking specifically at exercise in NASH, which reported an apparent stability of HTGC following 6 months of exercise 9. The difference between these studies is most

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likely explained by the greater sensitivity of 1H-MRS (as used in this study) vs. histological assessment of percentage of hepatocytes affected the small number of cases in the previous study and the well-controlled design of the present study. Our HTGC data suggests that exercise may hold therapeutic benefits for people with NASH.

While the reduction in HTGC with exercise is encouraging, the changes reported here should be placed in context of studies using a combination of weight loss and exercise in

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ACCEPTED MANUSCRIPT NAFLD, which see a relative reduction of between 42-81% in HTGC following a mean weight reduction of 4-14% 7. Weight loss and exercise yielding a 10% reduction in body weight can reverse NASH 6, 8, highlighting the summative benefits of weight loss and exercise. Although weight loss is undoubtedly effective in reducing HTGC, the difficulty

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of maintaining weight loss in the long-term is well documented 10. As such, the optimal

clinical value of exercise upon liver health for people with NASH appears likely to be an adjunct to caloric restriction. However, exercise without weight loss may represent an

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alternative therapy for patients who find weight loss difficult.

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The mechanisms underlying the change in HTGC following exercise in NASH reflect changes in energy balance, circulatory lipids and insulin sensitivity. Without any change in body weight, exercise reduced visceral fat by 12%. To date, only one study has assessed the effects of exercise in biopsy proven NASH 9, however this is the first RCT

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to assess the effects of exercise on HTGC, metabolic control, body composition alongside inflammatory and fibrosis markers in patients with NASH. Visceral fat is reported to be directly linked with liver inflammation and fibrosis, independent of insulin

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resistance and hepatic steatosis 20. The precise mechanism of how visceral fat applies its

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detrimental effects on liver metabolism, fibrotic and inflammatory consequences remain unclear, although influx of fatty acids and synthesis of cytokines and adipokines has been shown to promote liver lipid accumulation, insulin resistance and inflammation 20, 21. The present data supports the close relationship between visceral fat and HTGC, with baseline visceral fat correlating with HTGC and the change in visceral fat with exercise correlating with the change in HTGC.

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ACCEPTED MANUSCRIPT The apparent stability in metabolic control here is surprising given the reduction in HTGC and visceral fat, and the close link between HTGC, hepatic insulin sensitivity and endogenous glucose production 22, 23. Despite a 16% reduction in HTGC, 19 patients remained above the clinical 5% HTGC diagnosis, suggesting a larger reduction in HTGC

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is required to significantly improve hepatic insulin sensitivity and glucose control, as

recently demonstrated 24, 25. Irrespective of the effects of exercise upon glycemic control, the selective reduction of both visceral fat and HTGC with exercise, independent of

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weight loss, supports exercise as an adjunct therapy for NASH, as previously shown in

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NAFLD 11, 12.

In this study there was a reduction in CK-18, a marker of apoptosis and a defining pathological feature of NASH, known to correlate with liver damage and fibrosis 15. However, TNF-α, IL-6 and hsCRP all remained stable in the exercise group, despite

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changes in HTGC and visceral fat. Although elevated liver lipid is a prerequisite of NASH 26, dysregulated cytokine metabolism plays an important role in disease progression 4. Exercise studies in coronary artery disease, T2DM and NAFLD have

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reported reductions in circulating cytokines (IL-6 and TNF-α) 27, 28 and CK-18 29, raising

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the possibility of a protective effect of exercise. However, a combined weight loss and exercise programme in people with NASH also showed a stability in circulatory cytokines, with the exception of IL-6 which reduced 30. Importantly, circulatory inflammatory markers do not represent inflammation inside of the liver. As hepatic inflammatory markers were not directly assessed, HTGC may represent a better biomarker of free fatty acid flux, oxidative-, ER- stress that result in steatosis and progressive liver damage 31. Although there are clear biological links between

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ACCEPTED MANUSCRIPT inflammation and NASH, further studies are required to understand how lifestyle management may be optimised to address these.

Liver enzymes (ALT, AST) and non-invasive scores of liver disease (ALT/AST Ratio,

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NAFLD Fibrosis Score, Elevated Liver Fibrosis Test) did not change with 12 weeks of

exercise therapy. Furthermore, changes in HTGC were not correlated with liver enzymes or non-invasive scores of liver disease. Our study used liver enzymes and non-invasive

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measures of liver disease as follow-up biopsies to assess liver histology were not

ethically permitted. However, biopsy data does show that significant weight loss as a

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result of calorie restriction and exercise improves liver histology 6, 8. The lack of change in liver enzymes and biomarkers of liver disease following exercise suggest that exercise without weight loss may be insufficient to directly influence liver fibrosis. However, without a follow up biopsy it is difficult to truly ascertain the impact of exercise alone on

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NASH. The present data supports longer and larger studies investigating the differential and combined effects of exercise and weight loss in NASH to confirm their effects on

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Limitations

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disease progression.

The present study is not without limitation. Liver fibrosis was not measured directly from biopsy post-exercise. However, as recent work has suggested that steatosis and NASH may be more interchangeable than previously thought 2 it was ethically difficult to justify exposing volunteers to the risk of a repeat biopsy within such a short period of time. Although powered sufficiently for the primary outcome, the sample size may be insufficient to define secondary outcomes. The length of the study, although comparable

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ACCEPTED MANUSCRIPT with previous exercise studies in NAFLD, may not be long enough in duration to observe any improvements in histology and circulating inflammation in NASH.

Conclusion

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Exercise was well tolerated and reduced HTGC, visceral fat and circulating triglycerides in adults with biopsy proven NASH, independent of weight loss, but without any

apparent impact on circulating markers of inflammation or non-invasive scores of liver

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disease. These data suggest that increased exercise in the absence of weight loss is

effective at reducing HTGC, but may be less efficacious at ameliorating steatohepatitis

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over a 12 week programme. Clinically, exercise has a significant beneficial effect on HTGC and visceral fat. However, the optimal clinical value of exercise for people with NASH appears likely to be an adjunct to caloric restriction.

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Acknowledgements:

The authors would like to thank the patients for their time and enthusiasm and the funders

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for their support.

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References 1. Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol. 2013;10(6):330-44.. 2. McPherson S, Hardy T, Henderson E, et al. Evidence of NAFLD progression from steatosis to fibrosing-steatohepatitis using paired biopsies: Implications for prognosis and clinical management. Journal of hepatology. 2015;62(5):1148-55. 3. Singh S, Allen AM, Wang Z, et al. Fibrosis Progression in Nonalcoholic Fatty Liver vs Nonalcoholic Steatohepatitis: A Systematic Review and Meta-analysis of Paired-Biopsy Studies. Clin Gastroenterol Hepatol. 2015;13(4):643-54 e9. 4. Park EJ, Lee JH, Yu GY, et al. Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell. 2010;140(2):197-208. 5. Hardy T, Anstee QM, Day CP. Nonalcoholic fatty liver disease: new treatments. Curr Opin Gastroenterol. 2015;31(3):175-83. 6. Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, et al. Weight Loss via Lifestyle Modification Significantly Reduces Features of Nonalcoholic Steatohepatitis. Gastroenterology. 2015;149(2):367-78. 7. Thoma C, Day CP, Trenell MI. Lifestyle interventions for the treatment of nonalcoholic fatty liver disease in adults: a systematic review. Journal of hepatology. 2012;56(1):255-66. 8. Promrat K, Kleiner DE, Niemeier H, et al. Randomized controlled trial testing the effects of weight loss on nonalcoholic steatohepatitis. Hepatology. 2010;51(1):121-9. 9. Hickman I, J. , Byrne NM, Croci I, et al. Randomised study of the metabolic and histological effects of exercise in non alcoholic steatohepatitis. Journal of Diabetes and Metabolism. 2013;4(8). 10. Dudekula A, Rachakonda V, Shaik B, et al. Weight loss in nonalcoholic Fatty liver disease patients in an ambulatory care setting is largely unsuccessful but correlates with frequency of clinic visits. PloS one. 2014;9(11):e111808. 11. Johnson NA, Sachinwalla T, Walton DW, et al. Aerobic exercise training reduces hepatic and visceral lipids in obese individuals without weight loss. Hepatology. 2009;50(4):1105-12. 12. Hallsworth K, Fattakhova G, Hollingsworth KG, et al. Resistance exercise reduces liver fat and its mediators in non-alcoholic fatty liver disease independent of weight loss. Gut. 2011;60(9):1278-83. 13. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313-21. 14. McPherson S, Stewart SF, Henderson E, et al. Simple non-invasive fibrosis scoring systems can reliably exclude advanced fibrosis in patients with nonalcoholic fatty liver disease. Gut. 2010;59(9):1265-9. 15. Feldstein AE, Wieckowska A, Lopez AR, et al. Cytokeratin-18 fragment levels as noninvasive biomarkers for nonalcoholic steatohepatitis: a multicenter validation study. Hepatology. 2009;50(4):1072-8. 16. Biaggi RR, Vollman MW, Nies MA, et al. Comparison of air-displacement plethysmography with hydrostatic weighing and bioelectrical impedance analysis for the assessment of body composition in healthy adults. The American Journal of Clinical Nutrition. 1999;69(5):898-903.

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Longo R, Pollesello P, Ricci C, et al. Proton MR spectroscopy in quantitative in vivo determination of fat content in human liver steatosis. J Magn Reson Imaging. 1995;5(3):281-5. Hallsworth K, Thoma C, Hollingsworth KG, et al. Modified high-intensity interval training reduces liver fat and improves cardiac function in non-alcoholic fatty liver disease: A randomised controlled trial. Clin Sci (Lond). 2015;129(12):1097-105. Cassidy S, Thoma C, Hallsworth K, et al. High intensity intermittent exercise improves cardiac structure and function and reduces liver fat in patients with type 2 diabetes: a randomised controlled trial. Diabetologia. 2016;59(1):56-66. Van der Poorten D, Milner KL, Hui J, et al. Visceral fat: a key mediator of steatohepatitis in metabolic liver disease. Hepatology. 2008;48(2):449-57. Bergman RN, Kim SP, Catalano KJ, et al. Why visceral fat is bad: mechanisms of the metabolic syndrome. Obesity (Silver Spring). 2006;14 Suppl 1:16S-9S. Samuel VT, Liu ZX, Qu X, et al. Mechanism of hepatic insulin resistance in nonalcoholic fatty liver disease. J Biol Chem. 2004;279(31):32345-53. Seppala-Lindroos A, Vehkavaara S, Hakkinen AM, et al. Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity in normal men. J Clin Endocrinol Metab. 2002;87(7):3023-8. Cuthbertson DJ, Shojaee-Moradie F, Sprung VS, et al. Dissociation between exercise-induced reduction in liver fat and changes in hepatic and peripheral glucose homoeostasis in obese patients with non-alcoholic fatty liver disease. Clin Sci (Lond). 2016;130(2):93-104. Lim EL, Hollingsworth KG, Aribisala BS, et al. Reversal of type 2 diabetes: normalisation of beta cell function in association with decreased pancreas and liver triacylglycerol. Diabetologia. 2011;54(10):2506-14. Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998;114(4):842-5. Goldhammer E, Tanchilevitch A, Maor I, et al. Exercise training modulates cytokines activity in coronary heart disease patients. International journal of cardiology. 2005;100(1):93-9. Kadoglou NP, Perrea D, Iliadis F, et al. Exercise reduces resistin and inflammatory cytokines in patients with type 2 diabetes. Diabetes care. 2007;30(3):719-21. Fealy CE, Haus JM, Solomon TP, et al. Short-term exercise reduces markers of hepatocyte apoptosis in nonalcoholic fatty liver disease. J Appl Physiol (1985). 2012;113(1):1-6. Kugelmas M, Hill DB, Vivian B, et al. Cytokines and NASH: a pilot study of the effects of lifestyle modification and vitamin E. Hepatology. 2003;38(2):413-9. Ratziu V, Bellentani S, Cortez-Pinto H, et al. A position statement on NAFLD/NASH based on the EASL 2009 special conference. Journal of hepatology. 2010;53(2):372-84.

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Subject Characteristics

34 (5) 95 (9)

81 (59) 59 (27) 113 (78) 4.9 (1.3) 2.0 (0.9) 261 (83) 46.5 (2.5)

75 (52) 58 (30) 96 (53) 5.2 (1.3) 2.3 (1.0) 258 (75) 46.7 (3.3)

0.18 0.15 -

Baseline

0.28 0.33 0.08 0.07 0.02* 0.42 0.35

Time x Treatment Interaction p value

Posttreatment

p value

54 (12) 33 (7) 90 (18) 25 (8)

33 (7) 91 (18)

0.12 0.12

0.77 0.86

53 (25) 41 (14) 66 (46) 4.7 (1.4) 2.2 (1.0) 208 (42) 45.4 (3.8)

52 (18) 45 (12) 56 (33) 4.5 (1.3) 1.7 (0.8) 214 (44) 45.7 (3.4)

0.31 0.17 0.05* 0.26 0.09 0.06 0.31

0.44 0.30 0.82 0.20 0.03# 0.35 0.93

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51 (16) 33 (5) 94 (9) 21 (5)

p value

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Metabolic ALT (U/L-1) AST (U/L-1) GGT (U/L-1) Total Cholesterol (mmol/L) Triglyceride (mmol/L) Platelets (x10g/l) Albumin (g/L)

Posttreatment

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Anthropometry Age (years) BMI (kg/m-2) Weight (kg) VO2PEAK (mL/kg-1/min-1)

Baseline

Exercise (n=12)

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Table 1

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BMI, body mass index; VO2peak, aerobic capacity; ALT, alanine aminotransferase; AST, aspartate aminotransferase; gammaglutamyltransferase. Values are means (SD). * significant difference baseline versus post-intervention (p<0.05) # significant difference time x treatment interaction (p<0.05)

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Baseline liver histology and NAFLD fibrosis score Exercise (n=12) 5 (3-7)

Steatosis

1 (1-3)

2 (1-3)

Inflammation

1 (0-2)

2 (1-2)

Ballooning

1 (0-2)

1 (1-2)

Fibrosis Stage

3 (0-3)

0

1 (4%)

1

2 (8%)

2

3 (13%)

3

6 (25%)

4

0 (0%)

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0.47

0 (0%) 0 (0%)

5 (21%) 7 (29%) 0 (0%)

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Baseline liver histology and fibrosis stage is median (range) for all patients

0.61

0.80

3 (2-3)

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NAS

p value

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Control (n=12) 5 (2-7)

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Table 2

0.52

0.28

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Hepatic triglyceride content, adipose tissue, body composition and metabolic control Exercise (n=12)

Posttreatment

p value

Posttreatment

p value

p value

10 (5)

11 (5)

0.08

12 (9)

10 (6)

0.04*

0.02#

Visceral Adipose Tissue (cm²)

173 (75)

187 (53)

0.02*

191 (86)

169 (50)

0.04*

0.01##

Subcutaneous Adipose Tissue (cm²)

396 (124)

337 (181)

0.24

409 (113)

318 (158)

0.08

0.07

Fat Mass (kg)

38 (9)

38 (8)

0.37

35 (15)

33 (15)

0.07

0.24

Lean Body Mass (kg)

57 (7)

57 (7)

0.10

56 (10)

58 (10)

0.01**

0.51

Fasting Glucose (mmol/L)

5.8 (1.5)

5.8 (1.8)

0.41

6.7 (1.7)

6.6 (1.6)

0.38

0.80

Fasting Insulin (pmol/L)

98 (58)

91 (52)

0.18

118 (75)

99 (44)

0.23

0.96

HOMA-IR

1.6 (1.1)

1.7 (1.0)

0.18

2.3 (1.4)

1.9 (0.8)

0.26

0.53

HbA1c (mmol/mol)

47 (11)

49 (15)

0.16

52 (14)

50 (13)

0.14

0.13

879 (270)

0.23

1016 (279)

980 (329)

0.20

0.91

fsOGTT, AUC

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838 (191)

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Hepatic triglyceride content (%)

Baseline

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Baseline

Time x Treatment Interaction

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Control (n=12)

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Table 3

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HOMA-IR, homeostasis model of insulin resistance; HbA1c, glycated haemoglobin; fsOGTT, frequently sampled oral glucose tolerance test; AUC, area under the curve; Values are means (± SD). * significant difference baseline versus post-intervention (p<0.05) ** significant difference baseline versus post-intervention (p<0.01) # significant difference time x treatment interaction (p<0.05) ## significant difference time x treatment interaction (p<0.01)

ACCEPTED MANUSCRIPT

Liver Enzymes, NAFLD activity score, Elevated Liver Fibrosis Test and circulatory cytokines / inflammation. Exercise (n=12)

Time x Treatment Interaction

Post-treatment

p value

Baseline

Post-treatment

p value

p value

ALT/AST Ratio

0.92 (0.44)

0.93 (0.36)

0.42

0.86 (0.32)

0.92 (0.36)

0.10

0.63

NAFLD Fibrosis Score

-0.95 (1.43)

-0.98 (1.53)

0.36

-1.51 (1.00)

-1.50 (1.12)

0.47

0.80

Elevated Liver Fibrosis Test

8.8 (0.9)

8.8 (1.1)

0.46

9.4 (1.1)

9.5 (1.2)

0.35

0.76

TNF-α (pg/ml)

2.3 (0.7)

2.3 (0.7)

0.25

2.2 (0.6)

2.4 (0.7)

0.17

0.25

IL-6 (pg/ml)

2.4 (4.2)

2.0 (2.7)

0.38

1.4 (0.7)

1.7 (1.7)

0.44

0.77

hsCRP (mg/L)

2.1 (1.5)

3.1 (3.0)

0.48

2.8 (2.4)

4.9 (4.9)

0.21

0.71

842 (1140)

781 (1013)

0.44

627 (764)

441 (275)

0.43

0.82

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CK-18 (U/L)

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Control (n=12)

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Table 4

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ALT, alanine aminotransferase; AST, aspartate aminotransferase; NAFLD, non-alcoholic fatty liver disease; TNF-α, tumour necrosis factor alpha; IL-1ra, interleukin 1ra; IL-6, interleukin 6; IL-8, interleukin 8. Values are means (± SD).

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Enrollment

Assessed for eligibility (n= 31)

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Figure 1: CONSORT flow diagram.

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Excluded (n= 5)  Not meeting inclusion criteria (n= 4)  Declined to participate (n= 1)  Other reasons (n= 0)

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Randomized (n= 26)

Allocation

Allocated to exercise intervention (n= 13)  Received allocated intervention (n= 12) Did not receive allocated intervention (give reasons) (n= 1, due to knee problem)

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Allocated to standard care intervention (n= 13)  Received allocated intervention (n= 12) Did not receive allocated intervention (give reasons) (n= 1, due to back problem)

Follow-Up Lost to follow-up (give reasons) (n= 0

Discontinued intervention (give reasons) (n= 0)

Discontinued intervention (give reasons) (n= 0)

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Lost to follow-up (give reasons) (n= 0)

Analysed (n= 12)  Excluded from analysis (give reasons) (n= 0)

Analysis Analysed (n= 12)  Excluded from analysis (give reasons) (n= 0)

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Supplement 1: Clinical Trial Study Protocol

ACCEPTED MANUSCRIPT ISRCTN16070927 DOI 10.1186/ISRCTN16070927

Part 1 What is the purpose of the research project?

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Condition category: Nutritional, Metabolic, Endocrine Date applied: 08/01/2015 Date assigned: 09/01/2015 Last edited: 22/01/2015 Prospective/Retrospective: Retrospectively registered Overall trial status: Completed Recruitment status: No longer recruiting

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Exercise and non-alcoholic steatohepatitis

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Non-alcoholic fatty liver disease (NAFLD) spans a spectrum of liver conditions ranging from fatty liver, through to liver inflammation and fibrosis, cirrhosis and endstage liver disease. Recent information has shown that exercise may help people with NAFLD. Exercise may help reduce the amount of fat in the liver by: 1) increasing the ability of the body to burn fat 2) increasing the sensitivity of the body to food. Evidence also suggests that exercise may help to reduce active injury and inflammation in the liver.

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We aim to show the effect of exercise on the levels of fat, injury and inflammation in the liver and the sensitivity of the body to food. We will measure the fat in the liver using a magnetic resonance scanner and levels of injury and inflammation through simple blood tests. This research DOES NOT require any further biopsies – we use the results from the biopsy that you have already had in clinic to see whether you would be suitable to take part in this study.

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Understanding the relationship between exercise and liver injury is important in gaining acceptance of exercise in the management of NAFLD and avoiding excess weight gain. Why have I been chosen? You have been chosen because you have evidence of active injury and inflammation in your liver (called non-alcoholic steatohepatitis or NASH) on your liver biopsy and currently do not do any regular exercise. The project will involve up to 28 people. Do I have to take part? Your participation is purely voluntary and all results will be strictly anonymous. If you decide to take part, you are still free to withdraw at any time without giving reasons and without your medical care being affected. If you do decide to take part, you will be given this information sheet to keep and be asked to sign a consent form. What will the research project involve? Version 1.1 2 January 2013

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You will be asked to attend the Clinical Research Facility at the Royal Victoria Infirmary and Newcastle Magnetic Resonance Centre, on 5 occasions over 4 months ACCEPTED MANUSCRIPT to have your metabolism and liver fat checked. If you take part in the exercise group you will also be asked to attend exercise sessions 3 times a week for 12 weeks.

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Visit 1:After reading this information sheet and after having had time to make a decision and ask any questions to the researcher, you will be asked to sign the consent form saying that you would like to take part in this research study. You will be asked to complete a screening questionnaire. You will then do a cycling test. During this test you will cycle at the same pace but how hard you are cycling will increase every minute. You will keep on cycling until you decide to stop or until pedalling becomes difficult. Whilst you are cycling you will be asked to wear a facemask and a heart rate monitor. The exercise test will last between 10 - 15 minutes. At the end of the test you will feel tired but will recovery very quickly. Total visit time: 1hour

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Visit 2:The level of fat in your liver will be measured using magnetic resonance imaging (MRI). This DOES NOT involve a biopsy, is completely painless and does not have any dangers associated with the examination. Finally, you will be asked to drink a large sugary drink (350ml Lucozade). A cannula (small plastic tube) will be placed in your arm so that blood samples can be collected at regular intervals for 2 hours after you drink the sugary drink. Total visit time: 3hours

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Visit 3:The amount of fat and muscle in your body will be measured whilst you sit quietly on a chair for 5 minutes in a machine called the Bodpod. Whilst in the Bodpod we ask you to wear swimwear as the Bodpod measures your body composition by measuring how much air you displace – if you are wearing baggy clothing this can affect the results. You will then be asked to lie quietly on a bed whilst your resting metabolism is measured. After this, you will be asked to cycle for 60 minutes at an easy pace. You can watch the TV or listen to music whilst you cycle. A cannula will be placed in your arm so that blood samples can be collected at regular intervals during exercise. You will also be asked to wear a facemask and a heart rate monitor. You will be given a small activity monitor to wear for seven days. This monitor looks at the amount of calories you burn on a daily basis, how many steps you take and how generally active you are. Total visit time: 3hours You will be assigned to one of two groups. This allocation is random and is not controlled by the researchers. The first group will attend 3 exercise classes per week over 12 weeks. Each exercise session will involve you using a bike followed by weights. These sessions will be supervised by a clinical exercise specialist and the sessions will be individualised and will last about 45-60 minutes (like having a personal trainer). These sessions will be held at a sports centre that is convenient for you. It is important that your weight does not change over this time. You will be instructed to maintain your current weight throughout the study. The second group will not attend the exercise classes or be required to undertake any exercise over the 12 weeks.

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After 12 weeks, the metabolic evaluations performed at Visits 2 and 3 will be repeated, making 5 study visits in total.

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If you are placed in the group which does not attend any exercise sessions, at the end of the study you will be given the opportunity to have an individualised exercise programme, though this is not compulsory. No further tests will be undertaken if you choose to take part in the exercise. Expenses and payments

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Any travel / parking costs for helping with this research will be refunded. The research study will cover the costs for membership to the sports centre that you will attend if placed in the exercise group. What do I have to do?

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You will continue with your usual treatment(s) during the project. It will be important that you attend over 90% of the exercise sessions. Arrangements will be made for you to attend the research centres as necessary and there is parking available outside the centre, or if you wish a taxi will be arranged. You will be asked not to drink alcohol or exercise the day before the five test days. Each of the metabolic assessments will be in the morning before breakfast – you should not eat before you come to the centre. Food will be provided for you after the examination. What are the side effects of treatment received when taking part? This study involves exercise so there are no side effects (such as may occur with drugs). The exercise will be as safe as the researcher can make it but there may still be minimal risks.

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Are there any other possible disadvantages of taking part? Giving up time to participate has to be considered.

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What are the possible benefits of taking part?

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Being more physically active may be beneficial to the level of fat, injury and inflammation in your liver and if sustained after the study, may help in preventing other complications such as heart disease and diabetes. You will have supervised exercise sessions (like a personal trainer) which will teach you about your body, show you how to exercise correctly and help you become more physically fit. If you are placed in the group which does not attend any exercise sessions, at the end of the study you will be given the opportunity to have an individualised exercise programme, though this is not compulsory. No further tests will be undertaken if you choose to take part in the exercise. What happens at the end of the research project? At the end of the project, we shall be able to inform you of how the exercise affected your liver and metabolism. What if there is a problem?

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If you have any concern or complaint about any aspect of the study this will be dealt with immediately by Professor Trenell. Contact details for the primary researcher are given at the end of Part 1. ACCEPTED MANUSCRIPT Will my taking part in the project be kept confidential? All information obtained during the course of the research project will be kept strictly confidential. Your own GP and liver doctor will be informed of your participation in the project.

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What will happen to the results of the research study? The results of the project will be presented in national and international liver meetings and will be published in one of the liver journals. You will not be identified in any report or publication. You will be welcome to have a copy of the results once they are published.

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Who has reviewed the study?

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Ethical review of the study has been conducted by Sunderland Research Ethics Committee. Who are the contacts for further information? Further information can be obtained from:

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Thank you.

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Kate Hallsworth MoveLab; Physical Activity and Exercise Research Faculty of Medical Sciences 4th Floor William Leech Building Newcastle University Newcastle upon Tyne NE2 4HH Tel: 0191 208 8264 or email [email protected]

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Part 2 ACCEPTED MANUSCRIPT What if relevant new information becomes available? If new information is published during the course of a study this can sometimes change how the research should go forward. However, for this study it is most unlikely that this would occur. The study design is unique, and there are very few research groups worldwide able to carry out studies of this kind.

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What will happen if I don’t want to carry on with the study? You would be able to withdraw from the study at any time. Measurements already made would still be used if you were to agree to this. What if there is a problem?

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a) Complaints If you have any concern or complaint about any aspect of this study you should contact Professor M Trenell by phone on 0191 2226935, or write to him at the address at the end of Part 1 of this document. If you remain unhappy you can contact the Research Manager of the Newcastle upon Tyne Hospitals NHS Trust (Dr C Mackerness, Clinical Research Centre, RVI, Queen Victoria Road, Newcastle upon Tyne NE1 4LP; Tel 0191 282 5959).

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b) Harm In the unlikely event that something does go wrong and you suffer in any way, the arrangements are as follows: If negligence of staff led to harm, then this would be covered by the Newcastle upon Tyne Hospitals Trust clinical negligence scheme. You may have to meet legal costs. If any harm was non-negligent then the hospital trust may consider a discretionary payment. Will my taking part in the project be kept confidential?

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All information obtained during the course of the research project will be kept strictly confidential. This will be achieved by storing information in password-protected computer files, and appointment information in locked filing systems within the Magnetic Resonance Centre and Clinical Research Facility. No individually identifiable information will be stored outside the Centre. Analysis of the detailed results of the research will be done by Professor Trenell, Dr Hallsworth, Dr Hollingsworth, Dr Anstee, Mr Thoma and Professor Day. At this stage no personal information is part of the dataset. Results will be sent to participants, presented at scientific meetings and published in scientific journals without personal identification of any volunteer although thanks to the volunteers will be recorded. Your own GP will be informed of your help with this study, and this is normal practice. Your specialist liver consultant will also be aware. The detailed results of the research tests will not be sent to anybody outside the University.

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What will happen to blood samples?

ACCEPTED MANUSCRIPT The samples will be tested for liver function, insulin, sugar, fat, inflammation, fibrosis and other food derived substances. Samples will be stored until it is certain that the test results are accurate, and then they will be disposed of. During storage, samples are identified only by a code number, not your name. No other tests will be carried out on the samples. What will happen to results of the research?

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Who is organising and funding the research?

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The results will be presented at scientific meetings for discussion by other experts in this field. They will be written up in the form of a scientific paper and this will be intended to be published in a suitable scientific journal. As soon as the results are fully analysed after the end of the entire study you will receive a letter describing what we have found, and what implications it has for people with NASH. We will also hold an open evening for participants at which we will present the results of the study.

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This project is funded from a project grant from the Newcastle Hospitals Trust Joint Research Committee. The design and organisation of the study is the responsibility of Professor Trenell and Professor Day who are internationally recognised as experts in this field. There is no payment to any of the researchers involved in this study. They are employed by Newcastle University to work in the NHS, to teach and to research and have no financial link with the study. Who has reviewed the study?

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Ethical review of the study has been conducted by Sunderland Research Ethics Committee. Design of this information sheet

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This document is written in accordance with the requirements of the European Clinical Trials Directive 2001/20/EC, the ICH Good Clinical Practice guidelines and the UK Medicines for Human Use (Clinical Trials) Regulation 2004.

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ACCEPTED MANUSCRIPT 1

Supplement 2: Detailed Methods

2 Protocol

4

Twenty six sedentary (<60 minutes of moderate-vigorous activity per week) patients

5

were randomly assigned using a permuted blocks method with an independent

6

member of the research group 1 to either exercise intervention (n = 13) or standard

7

care (n = 13) (Figure 1). Participant characteristics are summarised in Table 1.

8

Following an initial screening visit, patients underwent a full medical history, physical

9

examination and progressive exercise test to screen for any undiagnosed cardiac

10

disease as previously described 21. Body composition, HTGC and abdominal fat

11

were measured using air displacement plethysmography and a 3.0 Tesla Philips

12

Achieva magnetic resonance image scanner respectively, also as previously

13

described 2-6.

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3

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14 Blood analyses:

16

A 75 g glucose load (Lucozade Original; Glaxo SmithKline, Brentford, UK) was then

17

consumed within 5 min and blood samples were then taken at 15, 30, 45, 60, 75, 90

18

and 120 minutes. Samples were analysed for whole blood glucose (YSI 2300 Stat

19

Plus-D, Yellow Springs Instruments, Yellow Springs, OH), and plasma insulin (Coat-

20

A-Count Insulin RIA kit, Diagnostic Products Corporation, CA, USA). Area under the

21

curve (AUC) for the resulting glucose response profile was calculated using the

22

trapezoidal rule 7 and insulin resistance determined using the homeostasis model

23

assessment (HOMA-IR) 8. Fasting samples were also analysed in a Clinical

24

Pathology Accredited laboratory (Newcastle Upon Tyne Hospital NHS Foundation

25

Trust, Department of Clinical Biochemistry) for: ALT, AST, GGT, total cholesterol,

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ACCEPTED MANUSCRIPT triglycerides and HbA1c. Total cholesterol, triglycerides, ALT, AST and GGT were

27

measured using a Roche Modular P test kit, CK-18 using M30-Apoptosense ELISA

28

kit (PEVIVA, Bromma, Sweden) 9, hsCRP (Roche Diagnostics Ltd, Burgess Hill, UK)

29

and HbA1c was measured using a TOSOH HLC-723G7 (Tosoh Corporation, Tokyo,

30

Japan). Plasma samples were analysed for circulatory inflammatory markers using

31

V-PLEX K15049D plate (Meso-Scale, Rockville, USA), and serum samples were

32

analysed for levels of tissue inhibitor of matrix metalloproteinase 1 (TIMP-1),

33

hyaluronic acid (HA) and aminoterminal peptide of pro-collagen III (P3NP) at an

34

independent Clinical Pathology Accredited laboratory (North Middlesex University

35

Hospital NHS Foundation Trust).

37

Exercise Intervention

38

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Exercise was supervised and performed three times per week on non-consecutive

40

days for 12 weeks, as we have successfully used for patients with NAFLD 10 and

41

Type 2 diabetes 11.The exercise programme consisted of aerobic (cycling) and

42

resistance training and is detailed in online Supplement 1. The cycling included a 5-

43

minute warm up and 3 intervals on a fixed bike for 2 minutes with 1-minute rest in

44

between. Exercise intensity was based on the 6-20 point Borg rating of perceived

45

exertion (RPE) with bike intervals corresponding to a RPE of 16-18 (‘Very Hard’) 12.

46

This was followed by a resistance exercise circuit that comprised of five exercises:

47

hip and knee extension, horizontal row, chest press, vertical row and knee extension

48

(Precor, Woodinville, USA). Patients were provided with a suitable weight for each

49

resistance exercise based on a RPE of 14-16 (‘hard’). RPE was used to guide

50

intensity for safety, time effectiveness, its translational use in clinical practice and its

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ACCEPTED MANUSCRIPT effectiveness at determining one repetition maximum 13, 14. Each session lasted

52

between 45-60 minutes. All sessions were conducted at an accredited sports centre

53

and supervised by a certified exercise specialist, who recorded progress to ensure

54

adherence and encouraged exercise progression through adding resistance on the

55

bike and increasing the weights lifted as able. This also helped to improve safety,

56

adherence, and the opportunity to resolve any problems. Standard care consisted of

57

volunteers continuing any prescription medication and going for regular monitoring of

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their condition(s) with their normal GP and/or consultant(s). Following 12 weeks of

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exercise all follow up tests were performed within 24 hours of the final exercise

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session.

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61 62

References

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3. 4.

5.

6.

7. 8.

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Beller EM, Gebski V, Keech AC. Randomisation in clinical trials. Med J Aust. 2002;177(10):565-7. Epub 2002/11/14. Biaggi RR, Vollman MW, Nies MA, et al. Comparison of air-displacement plethysmography with hydrostatic weighing and bioelectrical impedance analysis for the assessment of body composition in healthy adults. Am J Clin Nutr. 1999;69(5):898-903. Epub 1999/05/08. Fields DA, Higgins PB, Radley D. Air-displacement plethysmography: here to stay. Curr Opin Clin Nutr Metab Care. 2005;8(6):624-9. Epub 2005/10/06. Hallsworth K, Fattakhova G, Hollingsworth KG, et al. Resistance exercise reduces liver fat and its mediators in non-alcoholic fatty liver disease independent of weight loss. Gut. 2011;60(9):1278-83. Epub 2011/06/29. Longo R, Pollesello P, Ricci C, et al. Proton MR spectroscopy in quantitative in vivo determination of fat content in human liver steatosis. J Magn Reson Imaging. 1995;5(3):281-5. Epub 1995/05/01. Sardinha LB, Lohman TG, Teixeira PJ, et al. Comparison of air displacement plethysmography with dual-energy X-ray absorptiometry and 3 field methods for estimating body composition in middle-aged men. Am J Clin Nutr. 1998;68(4):78693. Epub 1998/10/15. Le Floch JP, Escuyer P, Baudin E, et al. Blood glucose area under the curve. Methodological aspects. Diabetes care. 1990;13(2):172-5. Epub 1990/02/01. Bloomgarden ZT. Measures of insulin sensitivity. Clin Lab Med. 2006;26(3):611-33, vi. Epub 2006/08/30.

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