the impact of mtor, tfeb and bid on non-alcoholic fatty liver disease and metabolic syndrome

199 Pages·2015·3.05 MB·English

by  Hao Zhang

THE IMPACT OF MTOR, TFEB AND BID ON NON-ALCOHOLIC
FATTY LIVER DISEASE AND METABOLIC SYNDROME

Hao Zhang

Submitted to the faculty of the University Graduate School
in partial fulfillment of the requirements
for the degree
Doctor of Philosophy
in the Department of Pathology and Laboratory Medicine
Indiana University
August, 2015
Accepted by the Graduate Faculty, Indiana University, in partial
fulfillment of the requirements for the degree of Doctor of Philosophy. 

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Xiao-Ming Yin, M.D., PhD, Chair

Doctoral Committee
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Naga P. Chalasani, M.D.

May 18, 2015
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Raymond L. Konger, M.D.

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Jill R. Murrell, PhD

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Dedication

To my parents, Dalu Zhang and Jie Gao, who always stay with me and give me
tremendous love. All I have and will accomplish are only possible due to their
support and sacrifice.

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Acknowledgments

First, I would like to extend my most sincere and deepest gratitude to my mentor,
Dr. Xiao-Ming Yin. It has been very fortunate of me and my honor to be able to
work under his supervision. Dr. Yin is an extremely busy clinician scientist and
undertaking administration responsibilities. However, he still has devoted huge
amount of time in training me to be a scientist. He has instructed me from every
single experiment design to each of word and grammar correction in manuscript
writing. As a mentor, he not only has guided me through the research projects,
but also provided great support for my future career development. Without Dr.
Yin’s care and support in the last five years, it is impossible for me to fulfill my
dream of being a physician scientist. Again, it is with deep gratitude that I say I
could not have asked for a better mentor. I will always remember his help and
kindness in this most important five years of my life.

I would also like to express my sincere gratitude to other research committee
members: Dr. Chalasani, Dr. Konger and Dr. Murrell. Thank you for your
guidance and suggestions in my thesis projects. My committee members also
have kindly helped me in my career development. They were always standing
with me whenever I need any help.

I also want to take this opportunity to express my heartfelt appreciation to
members in Dr. Yin lab. Dr. Khambu, who has stayed alongside with me for
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almost four years. Dr. Khambu not only has helped me tremendously in
improving my experimental techniques but also have celebrated with me on good
days and offered insightful advice in my bad days. It was my fortunate to work
with him. I would also like to thank Dr. Jane Chen, who guided me through
experiment when I first entered the lab. She have also helped me with all my
animal experiments. Without Dr. Chen, I would not have finished these two thesis
projects smoothly. I thank Dr. Yong Li for his virus and molecular biology support
for my projects; Dr. Changshun Yu for his computer and software support;
previous lab members Dr. Min Li and Dr. Xi Chen for their guidance and help in
my experiments.

Finally, I would like to thank my parents. They are the people who made me
where I am now. They are the people who always stay with me whenever I am in
frustration. I hope my accomplishment could make them proud.

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Hao Zhang

THE IMPACT OF MTOR, TFEB AND BID ON NON-ALCOHOLIC FATTY LIVER
DISEASE AND METABOLIC SYNDROME

Non-alcoholic fatty liver disease and metabolic syndrome induced by high
nutrient status have increasingly become a global health concern as it cause
multiple complications. The mTOR complex is central in regulating anabolic
reactions within cells under growth factors or under high nutrients stimulation.
Constitutive and persistent activation of mTOR can impair cellular functions. In
the first part of this study, we demonstrate a damping oscillation of mTOR activity
during a long-term treatment of high fat diet. TFEB translocation and lysosomal
enzyme activity also oscillate, but in an opposite direction. TFEB controls the
lysosomal activity, autophagic degradation and lipid metabolism. Overexpression
of wild type and mutant TFEB could inhibit NAFLD development in mice. In
addition, TFEB location in nucleus inversely correlates with NAFLD severity in
patients. mTOR activation under hypernutrition status suppresses TFEB
translocation, inhibits lysosomal functions and autophagic degradation of lipid
droplets. Inhibition of mTOR activity by rapamycin reverse the above
phenotypes. Because mTOR activation also requires normal lysosomal function,
the inhibition of TFEB by mTOR leads to decreased lysosomal function and
mTOR downregulation. This negative feedback may explain the oscillation
pattern of mTOR activation in long term high fat diet regimen and is a novel
mechanism for inhibition of mTOR. In the second part of study, we report that Bid
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protein, previously known for its pro-apoptosis function in promoting
mitochondrial permeability, plays an unexpected role in regulating fatty acid beta
oxidation. Deletion of Bid in mice reprograms the body’s response to hyper-
nutrition caused by high fat diet, leading to the resistance to the development of
obesity, liver steatosis and metabolic syndrome. These mice present a higher
oxygen consumption, a lower respiratory quotient, and an increased beta-
oxidation rate. Mechanistically, the high fat diet regimen triggers translocation of
the full length Bid molecule to mitochondrial membrane. Genetic deletion of Bid
also affects the stability of its binding protein, MTCH2 in the mitochondrial
membrane. In summary, we describe in this study a mTOR-TFEB-lysosome
feedback loop, which can regulate NAFLD development, and a novel Bid-
mediated regulatory mechanism in beta-oxidation, which limits energy
expenditure and promotes obesity development.

Xiao-Ming Yin, M.D., PhD, Chair

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Table of Contents

Part I. The Function of mTOR and TFEB in NAFLD and Obesity ......................... 1
1.1 Introduction ............................................................................................... 1
1.1.1 mTOR, the Pathway and The Regulation. ............................................ 1
1.1.1.1 Introduction ..................................................................................... 1
1.1.1.2 mTOR Complex and Upstream Regulators .................................... 2
1.1.1.3 Cellular Process Downstream of mTOR Signaling ......................... 7
1.1.2 mTOR in Lipid synthesis, Liver Function and Metabolism
Syndrome/NAFLD ........................................................................................ 10
1.1.2.1 mTOR and Lipid Homeostasis ...................................................... 10
1.1.2.2 Role of mTOR in Liver Metabolic Functions.. ................................ 13
1.1.3. TFEB-lysosome Regulation Pathway ................................................. 17
1.1.3.1 Introduction ................................................................................... 17
1.1.3.2 Regulation of TFEB ...................................................................... 18
1.2.3.3 Downstream Effects upon TFEB Activation .................................. 20
1.1.4. Autophagy, Its Regulation Pathways, and Functions in Liver ............ 24
1.1.4.1 Introduction ................................................................................... 24
1.1.4.2 The Molecular Mechanism of Autophagosome Biogenesis .......... 26
1.1.4.3 Regulation of Autophagy by Metabolic and Stress Pathways ....... 31
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1.1.4.4 Autophagy in Liver Diseases-General Introduction ....................... 33
1.1.4.5 Autophagy and Alcoholic/non-alcoholic Fatty Liver Disease ......... 36
1.2. Materials and Methods ............................................................................. 42
1.2.1 Animal Experiment .............................................................................. 42
1.2.2 Immunoblotting ................................................................................... 42
1.2.3 Immunostaining and Bodipy Staining .................................................. 43
1.2.4 Histology ............................................................................................. 44
1.2.5 ALT, Triglyceride and Cholesterol measurement ................................ 44
1.2.6 Hepatic Triglyceride Measurement ..................................................... 45
1.2.7 Lysosome Enzyme Activity Assay ....................................................... 45
1.2.8 Human Patient Study .......................................................................... 46
1.2.9 Gene Expression Analysis: Real-time PCR ........................................ 46
1.2.10 Statistical Analysis ............................................................................ 48
1.3 Results: ................................................................................................... 49
1.3.1 The mTOR Activity Oscillates Under the Sustained Hyper-nutrition
Condition. ..................................................................................................... 49
1.3.2 TFEB is Activated in an Oscillation Pattern Under the Sustained
Hyper-nutrition Condition. ............................................................................ 51
1.3.3. Lysosomal Functions Oscillate during Long-term High Fat Diet
Feeding. ....................................................................................................... 53
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1.3.4. Autophagy Degradation is Dynamically Regulated. ........................... 54
1.3.5. Degradation of Lipid Droplets by Lipophagy during NAFLD
Development. ............................................................................................... 56
1.3.6. The Subcellular Location of TFEB Location Correlates Highly with
NAFLD and Obesity in Human. .................................................................... 58
1.3.7. Suppression of mTOR Activity Promotes TFEB Translocation to the
Nucleus and Rescues the Lysosome Function in the Fatty Livers. .............. 60
1.3.8. Overexpression of Wild Type or mTOR resistant TFEB Mutant
Improves Lysosomal Functions, Hepatic Fat content and Liver Injury in
3wk High Fat Diet Mice. ............................................................................... 62
1.3.9. Overexpression of TFEB Improves Lysosomal Functions, Hepatic
Fat content and Liver Injury in 16wk High Fat Diet Fed Mice. ...................... 64
1.3.10. Consistent Activated RagA Mutant Inhibited TFEB Translocation,
TFEB Activity, Lysosomal Function and Liver Function. .............................. 65
1.4 Discussion: ................................................................................................ 67
1.5 Figures and Figure Legend ....................................................................... 71
Part II. The function of Bid in NAFLD and obesity .............................................. 97
1.1 Introduction ............................................................................................... 97
1.1.1 Bid and its function .............................................................................. 97
1.1.1.1 Introduction ................................................................................... 97
1.1.1.2 The Structure and Transcriptional Regulation of Bid .................... 98
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