DISSERTATION NOVEL THERAPIES FOR NAFLD Submitted by Angela Marie Nivala Department

148 Pages·2011·2.38 MB·English

DISSERTATION
NOVEL THERAPIES FOR NAFLD

Submitted by
Angela Marie Nivala
Department of Food Science and Human Nutrition

In partial fulfillment of the requirements
For the Degree of Doctor of Philosophy
Colorado State University
Fort Collins, Colorado
Spring 2011

Doctoral Committee:

Advisor: Michael Pagliassotti

Jorge Vivanco
Melinda Frye
Jairam Vanamala
Copyright by Angela Marie Nivala 2011
All Rights Reserved
ABSTRACT
NOVEL THERAPIES FOR NAFLD
BACKGROUND/AIMS
Non Alcoholic Fatty Liver Disease (NAFLD) is a chronic liver disease often associated with
metabolic disorders like type 2 diabetes, cardiovascular disease, obesity, and metabolic
syndrome. It is characterized by hepatic fat accumulation (steatosis) that is at or above
5% of liver weight in the absence of excessive alcohol consumption (< 20 g/day). Current
treatment for NAFLD focuses on reducing body weight and improving insulin action. The
intent of this thesis was to identify therapies that targeted the liver. In the first aim, we
examined whether secretions from plant roots contained compounds that restricted
lipid accumulation or improved insulin action. The second aim examined whether
taurine could prevent characteristic features of disease progression. Specifically, we
hypothesized that (1) onion root exudates will prevent or reduce lipid accumulation and
improve insulin signaling and (2) taurine will prevent or reduce endoplasmic reticulum
(ER) stress, oxidative stress and liver injury.

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METHODS
To examine these hypotheses, liver cells and dietary models of NAFLD were employed.
Analyses focused on hepatic triglycerides, insulin signaling, ER stress, oxidative stress
and inflammation using basic biochemical methods such as western blotting, Real Time
PCR, immunohistochemistry and enzyme-linked assays.

RESULTS
Onion root exudates prevented fatty acid-mediated lipid accumulation and
enhanced insulin signaling in H4IIE liver cells. Onion root exudates reduced plasma
glucose, free fatty acids (FFA), and improved insulin action in rats fed a high fat diet.
Taurine mitigated palmitate-mediated caspase-3 activity, cell death, ER stress,
and oxidative stress in H4IIE liver cells and primary hepatocytes. In rats fed a high
sucrose diet, taurine supplementation significantly reduced hepatic lipid accumulation,
liver injury, inflammation, plasma triglycerides and insulin levels. The high sucrose diet
resulted in an induction of multiple components of the unfolded protein response (UPR)
in the liver consistent with ER stress which was ameliorated by taurine
supplementation. Treatment of mice with the ER stress inducing agent tunicamycin
resulted in liver injury, UPR induction and hepatic lipid accumulation, and this was
significantly ameliorated by supplementation with taurine.

CONCLUSION
Both onion root exudates and taurine reduced metabolic abnormalities
associated with NAFLD. Onion root exudates appear to exert this effect through reduced
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lipid accumulation and enhanced insulin sensitivity in the liver, while taurine reduced
hepatic steatosis, ER stress, oxidative stress and liver injury. Overall, onion root
exudates and taurine show promise as novel therapies for the treatment of NAFLD.
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ACKNOWLEDGEMENTS
The author would like to thank her advisor, Dr. Michael Pagliassotti for all of the
support, generosity, encouragement, patience, and understanding throughout this
doctoral process. Thank you for providing such a great and nurturing environment that
has not only given me confidence as a scientist, but also hope that a career in science is
something worth striving for. “Leaving something better than how you found it” can
apply to people as well as things; my confidence, skills, and overall outlook on life has
changed profoundly for the better since becoming a part of your lab. I don’t have the
words to describe how grateful I am for this opportunity. Thank you to my committee
members Dr. Jorge Vivanco, Dr. Melinda Frye, and Dr. Jairam Vanamala for taking time
to participate and add valuable input into my development as a scientist. Your support
throughout this process is greatly appreciated. Thank you Dr. Yuren Wei and Dr. Dong
Wang for all that you do in the lab, especially in your contributions to the onion
exudates and taurine projects; this thesis would not exist if not for your amazing
abilities and hard work. Thank you Dr. Chris Gentile for all of your contributions,
especially in the taurine chapter. Your advice has been invaluable. Thank you Vivanco
Lab, Jennifer Kemp, Dr. Michael Bizeau, Kale Flory, and Jessica Stover for your
contributions to the onion exudates project. Thank you McLean Lab, Peterson Lab, Dr.
Kyle Pfaffenbach, and Jon Gonzalez for their contributions to the taurine project. Thank
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you Rebecca Cox and Lauren Reese for your help in the lab. Thank you to my husband
Peter for your mad formatting skills and all that you do, you are loved and appreciated
more than you know. Thank you to my daughter Lily, for not only reminding me to make
time for the important things in life but to never give up on them either.
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TABLE OF CONTENTS
CHAPTERS
Abstract ......................................................................................................... ii
Acknowledgements ....................................................................................... v
Table of Contents ......................................................................................... vii
Introduction ................................................................................................... 1
Plant Root Exudation .................................................................................................... 2
Taurine .......................................................................................................................... 3
Bibliography .................................................................................................................. 4
Literature Review ........................................................................................... 8
NAFLD ............................................................................................................................ 8
Etiology of NAFLD ......................................................................................................... 9
Consequences of Hepatic Steatosis ............................................................................ 13
Triggers Inducing Nash Development ......................................................................... 14
Oxidative Stress ........................................................................................................... 16
Cytokines/Adipokines ................................................................................................. 21
Bacterial Endotoxin ..................................................................................................... 24
ER Stress ...................................................................................................................... 25
Therapeutics ............................................................................................................... 30
Onion ........................................................................................................................... 31
Plant Root Exudation as a Source of Therapeutic Compounds .................................. 32
Taurine ........................................................................................................................ 34
Specific Aims ............................................................................................................... 37
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Bibliography ................................................................................................................ 38
Onion Plant Root Exudates Enhance Hepatic Insulin Signaling And Glucose
Homeostasis ................................................................................................ 59
Introduction ................................................................................................................ 59
Aim.... .......................................................................................................................... 61
Methods ...................................................................................................................... 61
Results ......................................................................................................................... 67
Discussion.................................................................................................................... 68
Bibliography ................................................................................................................ 75
Figure Legends ............................................................................................................ 82
The Protective Effects of Taurine on Nutrient-Induced Hepatic Endoplasmic
Stress, Oxidative Stress and Cell Damage .................................................... 87
Background ................................................................................................................. 87
Methods ...................................................................................................................... 90
Results ......................................................................................................................... 97
Discussion.................................................................................................................. 101
Bibliography .............................................................................................................. 106
Figure Legends .......................................................................................................... 116
Supplemental Data ................................................................................................... 127
Future Directions ....................................................................................... 131
Onion Exudates ......................................................................................................... 131
Taurine ...................................................................................................................... 135
Bibliography .............................................................................................................. 137
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CHAPTER 1
INTRODUCTION

Non Alcoholic Fatty Liver Disease (NAFLD) is an emerging disease associated with
metabolic dysfunction. NAFLD ranges from simple steatosis, or steatosis accompanied
by inflammation and fibrosis (steatohepatitis or NASH) that can further develop into
cirrhosis or hepatocellular carcinoma (HCC). The progression of simple steatosis to
NASH is thought to occur via the “two-hit hypothesis”;1 hepatic fat accumulation acts as
a predisposing factor (1st hit) to further insults (2nd hits) such as oxidative stress,
cytokines, bacterial endotoxin, or endoplasmic reticulum (ER) stress. In addition, fat
accumulation in the liver is associated with insulin resistant states, which is a common
feature among individuals with obesity, type 2 diabetes, and/or metabolic syndrome.2-10
In fact, NAFLD has been estimated to affect up to 30% of the population. There is a
substantial positive correlation between the rising obesity trend and NAFLD, which has
also been observed in obese children.2,11-15 There is no exclusive treatment for NAFLD,
and as a result it is often treated with therapies intended for obesity and diabetes.16
These treatments include weight loss, insulin sensitizers, lipid-lowering drugs, and/or
antioxidants, which have a number of side effects and are not designed to treat specific
characteristics of NAFLD such as hepatic steatosis, liver insulin resistance or the later
1