Prevalence and Severity of Non-alcoholic Fatty Liver Disease in a Large Prospective Primary Care

349 Pages·2014·7.34 MB·English

by  Matthew Armstrong

GLUCAGON-LIKE PEPTIDE-1 IN
NONALCOHOLIC STEATOHEPATITIS

By

Dr MATTHEW JAMES ARMSTRONG
MBChB(Hons) BSc(Hons) MRCP

A thesis submitted to the University of
Birmingham for the degree of
DOCTOR OF PHILOSOPHY

NIHR Liver Biomedical Research Unit
& Centre for Liver Research
School of Immunity and Infection
University of Birmingham
September 2013
University of Birmingham Research Archive

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ABSTRACT

Nonalcoholic fatty liver disease (NAFLD), and in particular its inflammatory component
steatohepatitis (NASH), are associated with significant risk of liver/cardiovascular morbidity
and death. My findings highlight that NAFLD is now the commonest cause of liver disease in
primary care, yet significant numbers with advanced fibrosis remain undetected. Application
of simple non-invasive scoring systems could aid with identifying those in greatest need of
intervention.

By adopting an integrative physiological approach with functional measures of lipid and
carbohydrate flux, I demonstrated that patients with NASH (vs. healthy controls) have
marked adipose tissue dysfunction (especially in abdominal subcutaneous adipose tissue),
alongside increased hepatic and muscle insulin resistance (IR). Targeting adipose-derived
lipotoxicity should be the mainstay of therapy in NASH. Glucagon-like peptide-1 (GLP-1)
based therapy (liraglutide) appears to be safe and well tolerated in patients at risk of
underlying NAFLD. My prospective randomised-controlled study highlighted that liraglutide
reduces metabolic dysfunction, hepatic lipogenesis, hepatic/adipose IR and inflammation in
patients with NASH. My in vitro studies in human hepatocytes indicate that the anti-steatotic
effects are not solely reliant on improvements in weight and/or glycaemic control. Taken
together, my findings highlight that GLP-1 based therapies have all the metabolic and clinical
attributes to make them a promising therapeutic option in patients with NASH. However,
the safety and histological efficacy of such awaits the completion of my 48-week Phase II
‘LEAN’ trial, which is integral as to whether larger clinical trials are warranted.
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DEDICATION

My thesis is dedicated to my beautiful wife Caroline, my long suffering parents Barbara and
John, and my big sis Emma. I would not be where I am today without their endless support
and devotion.

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ACKNOWLEDGMENTS

The work presented here would not have been possible without the dedication and
expertise of many friends and work colleagues over the last 4 years. In particular, I would
like to express my deep gratitude to my supervisors, Professor Philip Newsome and
Professor Jeremy Tomlinson, who have provided continuous support, guidance and
friendship throughout the whole of my time in Birmingham. I would like to also thank
Professor Stephen Gough for his expertise in industrial collaboration and guidance
throughout the clinical trial design.

Many individuals in the National Institute of Health Research (NIHR) liver biomedical
research unit/early development clinical trials team have provided practical help and
support with my clinical trial. Specifically, I would like to acknowledge Darren Barton, Piers
Gaunt, Kathy Guo and in particular Diana Hull for their tireless contributions and dedication
to the clinical trial, which at times feels like a thankless task. In addition, I would like to thank
the histopathology expertise of Professor Stefan Hübscher and Rachel Brown who continue
to work in their personal time to see that the clinical trial is a success. I would also like to
express my gratitude to the nursing staff and admin staff at the state-of-the-art Wellcome
Trust Clinical Research Facility and UHB liver outpatients department for their patience and
understanding throughout the trial. In addition, Bodil Elbrönd and Anders Toft of Novo
Nordisk Ltd were instrumental in my meta-analysis and in supporting my ideas and vision. I
would also like to thank the members of the BALLETS study team, including Louise Bentham,
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Alan Girling, Professor James Neuberger and in particular Professor Richard Lilford for our
insightful conversations and for allowing me to be part of their primary care projects.

I would also like to thank those at the NIHR Centre for Liver Research and Centre for
Diabetes, Endocrinology and Metabolism, whose collaborative efforts have enabled me to
learn new experimental techniques, which have formed the foundations of my in vitro work.
I would like to give specific mention to Maryam Nasiri, Laura Gathercole, Ricky Bhogal,
Jonathan Hazlehurst, Stefan Amisten (Oxford University) and Jinglei Yu for their practical
help and support. Several other scientists have given me valuable tips and encouragement
along the way, of which there are too many to mention. Specific mention must go, however,
to my close friend Laurence Hopkins for his insightful critique and comments along the way.
In addition, I would like to thank the support of my close friends and their families, who I
have been fortunate to work alongside in the last 4 years. In particular, I would like to thank
the Houlihan, Rowe, Parker, and Corbett families and Barney/Shankar for keeping me sane
and fed along the way.

Finally I would to thank the funders of the work presented here. These include a Clinical
Research Training Fellowship from the Wellcome Trust, study grant and free drug supplies
from Novo Nordisk Ltd and financial backing by the NIHR. In particular, I would like to
acknowledge the impressive infrastructure of Birmingham’s NIHR liver biomedical research
unit, which is largely due to years of dedication from Professor David Adams and his team;
without which none of this work would have been possible.

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TABLE OF CONTENTS
1 CHAPTER 1: GENERAL INTRODUCTION ............................................................................... 1
1.1 Nonalcoholic fatty liver disease (NAFLD) ............................................................... 1
1.1.1 Definition of NAFLD ........................................................................................................... 2
1.1.2 Diagnosis and staging of NAFLD ......................................................................................... 3
1.1.3 Epidemiology of NAFLD.................................................................................................... 16
1.1.4 Pathogenesis of NAFLD .................................................................................................... 18
1.1.5 Natural history and complications of NAFLD ................................................................... 25
1.1.6 Management of NAFLD .................................................................................................... 27
1.2 Glucagon-like peptide-1 (GLP-1) .......................................................................... 35
1.2.1 Physiology and actions of GLP-1 ...................................................................................... 35
1.2.2 The GLP-1 receptor (GLP-1R) ........................................................................................... 38
1.2.3 Development of GLP-1-based therapies for human use ................................................. 42
1.2.4 Exenatide (Byetta; Amylin Pharmaceuticals) ................................................................ 45
1.2.5 Liraglutide (Victoza; Novo Nordisk A/S) ........................................................................ 48
1.2.6 Potential for GLP-1 based therapies in NAFLD ................................................................ 62
1.3 Summary ............................................................................................................ 76
1.4 Aims of the project ............................................................................................. 77
2 CHAPTER 2: PRESENCE AND SEVERITY OF NONALCOHOLIC FATTY LIVER DISEASE IN A
LARGE PROSPECTIVE PRIMARY CARE COHORT ................................................................. 78
2.1 Introduction ....................................................................................................... 78
2.2 Methods ............................................................................................................. 80
2.2.1 Study population .............................................................................................................. 80
2.2.2 Data Definitions ............................................................................................................... 83
2.2.3 NAFLD Fibrosis Score (NFS) .............................................................................................. 84
2.2.4 Statistical Analysis ............................................................................................................ 85
2.3 Results................................................................................................................ 86
2.3.1 Causes of abnormal LFTs.................................................................................................. 87
2.3.2 Disease severity in the cohort of patients with NAFLD ................................................... 90
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2.4 Discussion .......................................................................................................... 92
3 CHAPTER 3: SAFETY AND EFFICACY OF LIRAGLUTIDE IN TYPE 2 DIABETIC PATIENTS WITH
ELEVATED LIVER ENZYMES: INDIVIDUAL PATIENT DATA META-ANALYSIS OF THE LEAD
PROGRAM ....................................................................................................................... 98
3.1 Introduction ....................................................................................................... 98
3.2 Methods ........................................................................................................... 101
3.2.1 Study population and design: ........................................................................................ 101
3.2.2 Data collection and definitions ...................................................................................... 103
3.2.3 Safety Profile .................................................................................................................. 104
3.2.4 Statistical Analysis .......................................................................................................... 104
3.3 Results.............................................................................................................. 107
3.3.1 Baseline demographics – meta-analysis ........................................................................ 107
3.3.2 Safety Profile – meta-analysis ........................................................................................ 109
3.3.3 Change in ALT – meta-analysis....................................................................................... 109
3.3.4 Change in Hepatic Steatosis – LEAD-2 sub-study .......................................................... 111
3.4 Discussion ........................................................................................................ 113
4 CHAPTER 4: LIRAGLUTIDE EFFICACY AND ACTION IN NONALCOHOLIC STEATOHEPATITIS
(LEAN): STUDY PROTOCOL FOR A PHASE II MULTI-CENTRE, DOUBLE-BLINDED
RANDOMISED-CONTROLLED TRIAL ................................................................................ 117
4.1 Introduction ..................................................................................................... 117
4.2 Methods ........................................................................................................... 120
4.2.1 Study Design Overview .................................................................................................. 120
4.2.2 Ethical and regulatory approval ..................................................................................... 121
4.2.3 Treatment groups .......................................................................................................... 122
4.2.4 Outcome Measures ........................................................................................................ 126
4.2.5 Analytical Methods ........................................................................................................ 127
4.2.6 Statistical Justification and Outcome Analysis ............................................................... 134
4.3 Conduct of the trial ........................................................................................... 137
4.3.1 Patient Selection ............................................................................................................ 137
4.3.2 Randomisation ............................................................................................................... 141
4.3.3 Medication preparation and blinding/unblinding procedures ...................................... 141
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4.3.4 Adverse event (AE) reporting and analysis .................................................................... 142
4.3.5 Study visit overview ....................................................................................................... 143
4.3.6 Storage of trial samples ................................................................................................. 145
4.3.7 Treatment compliance ................................................................................................... 146
4.3.8 Data handling, quality assurance, record keeping and retention.................................. 146
4.3.9 Case Report Forms ......................................................................................................... 147
4.3.10 Sponsorship, Indemnity and Monitoring ..................................................................... 147
4.3.11 Sources of funding ....................................................................................................... 147
4.4 Trial status ........................................................................................................ 148
4.5 Discussion ........................................................................................................ 149
4.5.1 Challenges in trial design ............................................................................................... 149
4.5.2 Safety profile of liraglutide ............................................................................................ 151
4.5.3 Summary ........................................................................................................................ 152
5 CHAPTER 5: ABDOMINAL SUBCUTANEOUS ADIPOSE TISSUE INSULIN RESISTANCE AND
LIPOLYSIS IN NONALCOHOLIC STEATOHEPATITIS ............................................................ 153
5.1 Introduction ..................................................................................................... 153
5.2 Methods ........................................................................................................... 156
5.2.1 Study subjects ................................................................................................................ 156
5.2.2 Study design ................................................................................................................... 157
5.2.3 Data Collection and Analysis .......................................................................................... 161
5.2.4 Statistical analysis .......................................................................................................... 165
5.3 Results.............................................................................................................. 166
5.3.1 Participant characteristics.............................................................................................. 166
5.3.2 Systemic IR ..................................................................................................................... 166
5.3.3 Hepatic IR ....................................................................................................................... 169
5.3.4 Hepatic DNL ................................................................................................................... 169
5.3.5 Depot-specific adipose tissue IR .................................................................................... 169
5.3.6 Serum adipocytokines and inflammatory cytokines ...................................................... 175
5.4 Discussion ........................................................................................................ 178
6 CHAPTER 6: LIRAGLUTIDE REDUCES ADIPOSE INSULIN RESISTANCE AND HEPATIC DE NOVO
LIPOGENESIS IN NONALCOHOLIC STEATOHEPATITIS: SUB-STUDY RESULTS OF A
RANDOMISED-CONTROLLED TRIAL (LEAN). .................................................................... 183
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6.1 Introduction ..................................................................................................... 183
6.2 Methods ........................................................................................................... 186
6.2.1 Study subjects ................................................................................................................ 186
6.2.2 Study design ................................................................................................................... 188
6.2.3 Data Collection and Analysis .......................................................................................... 189
6.2.4 Statistical analysis .......................................................................................................... 190
6.3 Results.............................................................................................................. 192
6.3.1 Study participants .......................................................................................................... 192
6.3.2 Clinical and metabolic variables..................................................................................... 194
6.3.3 Systemic insulin sensitivity ............................................................................................ 196
6.3.4 Hepatic insulin sensitivity .............................................................................................. 199
6.3.5 Hepatic DNL ................................................................................................................... 201
6.3.6 Adipose insulin sensitivity and lipolysis ......................................................................... 202
6.3.7 Serum adipocytokines and inflammatory mediators .................................................... 207
6.4 Discussion ........................................................................................................ 209
7 CHAPTER 7: DIRECT EFFECT OF GLP-1 ON THE LIVER IN-VITRO ........................................ 217
7.1 Introduction ..................................................................................................... 217
7.2 Methods ........................................................................................................... 220
7.2.1 Presence of GLP-1 receptor on liver cell types .............................................................. 220
7.2.2 Direct effect of GLP-1 analogues in human hepatocytes .............................................. 227
7.2.3 Statistical Analysis .......................................................................................................... 240
7.3 Results.............................................................................................................. 241
7.3.1 GLP-1R expression on liver cell types ............................................................................ 241
7.3.2 GLP-1 effect on Huh 7 cell viability ................................................................................ 244
7.3.3 GLP-1 effect on G-protein coupled receptor signalling ................................................. 246
7.3.4 Anti-steatotic effect of GLP-1 ........................................................................................ 247
7.3.5 GLP-1 effect on DNL in hepatocytes .............................................................................. 249
7.3.6 GLP-1 effect on NEFA uptake and -oxidation .............................................................. 249
7.4 Discussion ........................................................................................................ 253
8 CHAPTER 8: GENERAL DISCUSSION AND CONCLUDING REMARKS ................................... 260
8.1 Clinical burden of NAFLD and advanced fibrosis in primary care ........................ 260
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