The contribution of vascular adhesion protein-1 to glucose and lipid homeostasis in the liver

390 Pages·2013·9.09 MB·English

by  Sumera Karim

THE CONTRIBUTION OF VASCULAR ADHESION PROTEIN-1 TO GLUCOSE
AND LIPID HOMEOSTASIS IN THE LIVER

BY

SUMERA KARIM

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

Institute of Biomedical Research
The School of Immunity & Infection
College of Medical & Dental Sciences
University of Birmingham
May 2013
University of Birmingham Research Archive

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ABSTRACT
NAFLD is characterized by simple steatosis, which can progress to chronic inflammation and
fibrosis. Vascular Adhesion Protein-1 (VAP-1) is an adhesion molecule with semicarbazide-
sensitive amine oxidase (SSAO) activity, which is also expressed as a soluble protein in
serum (sVAP-1) and elevated in inflammatory liver diseases such as NAFLD. VAP-1 has
been shown to modulate glucose and lipid uptake in muscle and adipose tissue and thus we
investigated whether it may contribute to glucose and lipid homeostasis in human liver tissue.
Recreating the multicellular liver environment using an ex-vivo model we have shown that
activation of VAP-1 by its substrate methylamine leads to activation of NF-κB, glucose
uptake and lipid accumulation in the human liver with changes in transporter expression of
GLUT4, GLUT10 and GLUT13, as well as FABP2, LRP1, FATP2, FATP3, FATP4 and
FATP6. We have also documented changes in transporter expression profiles in human
disease. In conclusion, we demonstrate for the first time global alterations in cellular
expression of glucose and lipid transporter proteins in NASH. We confirm that VAP-1 is
elevated in disease and that SSAO activity of VAP-1 results in enhanced hepatic glucose and
lipid accumulation with changes in transporter expression. Thus we propose that bioactive
metabolites of SSAO activity contribute to the metabolic derangement evident in fatty liver
disease.
Work arising from this thesis

Published Manuscripts

An in vitro model of human acute ethanol exposure that incorporates CXCR3 and
CXCR4 dependent recruitment of immune cells,
Sumera Karim, Evaggelia Liaskou, Samuel Hadley, Janine Youster, Jeffrey Faint, David H
Adams, Patricia F Lalor, Toxicological Sciences, January 2013 132 (1): 131-141

Hepatic expression and cellular distribution of the glucose transporter family, Sumera
Karim, David H Adams, Patricia F Lalor, World Journal of Gastroenterology, 2012
December 14; 18 (46): 6771-6781

Published Abstracts

Digestive Disorders Federation (DDF) combined meeting for BSG, AUGIS, BAPEN and
BASL. Joint Meeting, June 7–20 2012, Arena and Convention Centre (ACC), Liverpool,
UK. Poster presentation. (Awarded poster of distinction), published in Gut 2012; 61:Suppl
2 A86-A87.

The liver meeting, 62nd annual meeting of the American association for the study of
liver diseases (ASSLD), November 4-8 2011, Moscone West Convention Center, San
Francisco, CA. Poster presentation. Published in Hepatology, 2011, Volume 54, Number 4,
1174A.

British association for the study of liver (BASL), 7-9 September 2011, Royal College of
physicians, London. Poster presentation. Published in special addition of GUT 2011; 60:
Supplement 2, A40-A41

American society for Mass Spectrometry, 59th Annual Meeting. June 5-9th 2011, Denver,
Colorado. (Poster presentation). Published in Journal for the American Society for Mass
Spectrometry, Volume 22, Supplement 1.

International presentations

International society for Hepatic Sinusoidal Research, 16th International Symposium on
cells of the Hepatic Sinusoid, Sinusoidal cells and the metabolic syndrome. September 22-
24, 2011. Florence, Italy.
Manuscripts In preparation

Dysregulated hepatic expression of glucose transporters in NASH: The contribution of
amine oxidase activity to hepatic glucose uptake.
Sumera Karim, Evaggelia Liaskou, Janine Fear, Abhilok Garg, Lee Claridge, Gary
Reynolds, Sirpa Jalkanen, David H Adams, Philip N. Newsome and Patricia F Lalor.

Use of MALDI Image analysis to determine disease-specific expression of variant forms
of L-FABP in non-alcoholic fatty liver disease.
Josephine Bunch, Sumera Karim, Joscelyn Sarsby, Anna Bradford, Evangelia Liaskou,
Janine Fear, Patricia F. Lalor.

The formation of atheroma is linked to steatohepatitis and hyper triglyceridaemia by
inflammatory signalling through the lymphotoxin-β receptor.
Mathew J. Harrison, Cécile Bénézech, Sumera Karim, Carl F. Ware, Patricia. F. Lalor, Jorge
H. Caamaño, G. Ed Rainger.

Multivariate and correlation analysis of MALDI mass spectrometry imaging data
reveals features in human Non-Alcoholic Steatohepatitis (NASH) liver samples.
Andrew D. Palmer, Alan M. Race, Rian L. Griffiths, Rory T. Steven, Sumera Karim,
Patricia F Lalor, Iain Styles, Josephine Bunch.
Learn from yesterday, live for today, hope for tomorrow. The important thing is not to stop
questioning.

Albert Einstein
Dedication

I dedicate this thesis to mum and my late father Abdul Karim.
ACKNOWLEDGEMENTS

I would like to take this opportunity and show my gratitude to my supervisor Dr PF Lalor and
co supervisor Professor P Newsome who gave me the opportunity to do this PhD and also to
Professor DH Adams. In particular I would like to say a special thank you to Dr PF Lalor for
her excellent guidance throughout my PhD and for supporting me not only academically but
also personally. I am eternally grateful to her. Thank you also to my past and present
industrial supervisors for their support and advice on microarrays.

This PhD would not have been possible without my funding bodies, so a special thank you to
BBSRC and my industrial sponsors for funding my PhD. Professor S Jalkanen for providing
VAP-1 constructs and our collaborators from chemistry Dr J Bunch and Rian Griffiths. A big
thank you to everyone in the liver labs for all their support in particular Ben James and Dr
Evangelia Liaskou who showed me many of the techniques when I started and Dr Lee
Claridge for providing the mouse samples used in this study.

I feel I have had an amazing journey both scientifically and personally and although there are
many lows and highs during a PhD the highs certainly make up for the lows. Last but not
least I would like to thank my family, firstly my mum who has always been there for me, who
has never let me down and who’s supported me in everything I have ever done. I would like
to thank my husband and my best friend Naveed Liaquat who has believed in me and has
supported me and has always found a way to cheer me up, to the rest of my family thank you
for your continued support and encouragement and finally thank you to my beautiful son
Ismaeel Sabih Naveed for being a good little boy whilst mummy was writing up.
TABLE OF CONTENTS

CHAPTER 1: GENERAL INTRODUCTION ...................................................................... 1
1.1 Overview ...................................................................................................................... 2
1.1.1 The Human Liver ................................................................................................... 2
1.1.2 The Liver Acinus .................................................................................................... 3
1.1.3 Liver cells ............................................................................................................... 5
1.1.4 Functions of the liver ............................................................................................ 10
1.1.5 The role of the liver in glucose homeostasis ........................................................ 10
1.1.6 The role of carbohydrate transporters in glucose metabolism ............................. 15
1.1.7 The role of the liver in lipid homeostasis ............................................................. 16
1.1.8 The role of fatty acid transporters in lipid metabolism ........................................ 18
1.1.9 Obesity and the Metabolic Syndrome .................................................................. 20
1.1.10 Non-Alcoholic Fatty Liver Disease ...................................................................... 21
1.1.11 Vascular Adhesion Protein- 1 .............................................................................. 30
1.1.12 VAP-1 is a multifunctional protein ...................................................................... 32
CHAPTER 2: MATERIALS & METHODS ....................................................................... 36
2.1 Human Tissue .............................................................................................................. 37
2.2 Murine Tissue .............................................................................................................. 37
2.3 Generation of Precision-Cut Liver Slices (PCLS) ...................................................... 38
2.4 Preparation of tissue sections for immunochemistry .................................................. 40
2.5 Sectioning of liver tissues for Matrix Assisted Laser Desorption Ionization Mass
Spectrometry Imaging (MALDI-MSI) ................................................................................. 40
2.6 Immunohistochemical staining of human liver specimens ......................................... 40
2.6.1 Immunofluorescent staining ................................................................................. 41
2.7 Histological staining to investigate liver morphology, steatosis and fibrosis ............. 43
2.8 Cell Isolation, Culture and Maintenance ..................................................................... 45
2.8.1 Isolation and culture of HSEC, BEC and Fibroblasts from human liver tissue ... 45
2.8.2 Isolation and culture of primary hepatocytes ....................................................... 47
2.8.3 Isolation and culture of peripheral blood mononuclear cells (PBMC) ................ 48
2.9 Culture and maintenance of hepatocyte cell lines ....................................................... 48
2.10 Maintenance of cells by freezing and storing ........................................................... 49
2.11 Adenoviral Transfection of Human HSEC with VAP-1 Constructs ......................... 49
2.12 Single Color Flow Cytometry ................................................................................... 50
2.13 Determination of cell and PCLS function and viability in culture ........................... 51
2.13.1 Treatment of cells and PCLS with palmitic and oleic acid .................................. 51
2.13.2 Quantification of lipid accumulation in fatty acid treated cells and PCLS using
ORO labelling ................................................................................................................... 52
2.13.3 Use of MTT to assess viability of PCLS and cultured cells ................................. 52
2.14 Assessment of lipid accumalation in PCLS and hepatic cells after VAP-1 treatment
53
2.15 Assessment of triglyceride secretion from PCLS after VAP-1 interventions ........... 54
2.16 Assessment of glucose uptake in PCLS and hepatic cells after VAP-1 treatment
using 2- Deoxy-D- Glucose [3H (G)] uptake assay ............................................................. 54
2.17 Validation of Hoechst dye for cell quantification ..................................................... 55
2.18 Determination of PCLS function and viability in culture ......................................... 56
2.18.1 Quantification of Albumin production for the assessment of hepatocyte synthetic
function in PCLS .............................................................................................................. 56
2.18.2 Assessment of glycogen storage in PCLS ............................................................ 57
2.18.3 Assessment of nuclear integrity in PCLS using Hoechst dye .............................. 58
2.19 Determination of the enzymatic activity of VAP-1 in whole tissues by the Amplex
Ultra Red method ................................................................................................................. 58
2.20 Analysis of gene expression in cultured cells and whole liver using microarrays and
quantitative PCR (qPCR) ..................................................................................................... 60
2.20.1 RNA extraction .................................................................................................... 60
2.20.2 cDNA synthesis .................................................................................................... 62
2.20.3 Microarray Analysis ............................................................................................. 62
2.20.4 Quantitative real-time PCR .................................................................................. 64
2.21 Whole tissue lipid analysis using Mass Spectrometry (MS) and Gas
Chromatography (GC) .......................................................................................................... 67
2.22 Extraction of lipids from liver tissue and PCLS for analysis by GC and MALDI-MS
dried droplet analysis ............................................................................................................ 67
2.23 Identification of lipid species in tissue extracts using MALDI-MS dried droplet
analysis ................................................................................................................................. 68
2.24 Identification and localization of lipids in tissue extracts using MALDI imaging ... 69
2.25 Statistical Analysis .................................................................................................... 70
CHAPTER 3: HEPATOCELLULAR EXPRESSION OF GLUCOSE AND LIPID
TRANSPORTERS .................................................................................................................. 71
3.1 Introduction ............................................................................................................... 72
3.1.1 Hepatic expression and function of carbohydrate transporters ............................ 73
3.1.2 Hepatic expression and function of lipid transporters .......................................... 80
3.2 Methods ...................................................................................................................... 89
3.3 Results ........................................................................................................................ 90
3.3.1 Microarray analysis reveals expression of GLUT receptors in human liver ........ 90
3.3.2 qPCR confirms expression of GLUT family receptors in human liver ................ 93
3.3.3 Liver epithelial cells express a varied GLUT receptor profile ............................. 96
3.3.4 GLUT receptor profile in non-epithelial cells ...................................................... 98
3.3.5 Immunohistochemical detection of GLUT protein expression in normal and
diseased livers ................................................................................................................. 100
3.3.6 Microarray analysis reveals expression of lipid trafficking proteins in human
liver 110
3.3.7 qPCR confirms expression of FABP receptors in human liver .......................... 114
3.3.8 Liver epithelial cells express FABP ................................................................... 117
3.3.9 FABP receptor profile in non-epithelial cells .................................................... 119
3.3.10 Immunohistochemical detection of fatty acid trafficking protein expression in
normal and diseased livers. ............................................................................................. 121
3.3.11 qPCR confirms expression of FATPs in human liver ........................................ 126
3.3.12 Liver epithelial cells express FATP receptors .................................................... 128
3.3.13 Liver non-epithelial cells express FATP receptors ............................................ 130
3.4 Discussion ................................................................................................................. 132
3.4.1 Hepatocellular expression of GLUT proteins .................................................... 134
3.4.2 Hepatocellular expression of fatty acid trafficking proteins .............................. 145
3.4.3 Mechanisms explaining changes in lipid transporter proteins ........................... 156
3.4.4 Future work ........................................................................................................ 158
CHAPTER 4: IS THERE A ROLE FOR VAP-1 IN GLUCOSE HOMEOSTASIS IN
THE LIVER? ........................................................................................................................ 162