Metabolic Abnormalities and Adipose Tissue Leukocyte Dynamics in a Murine Model of Obesity

155 Pages·2017·7.32 MB·English

Metabolic Abnormalities and Adipose Tissue Leukocyte Dynamics in a Murine
Model of Obesity, Weight Loss, and Weight Regain
By
Brian Francis Zamarron

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Immunology)
in the University of Michigan
2017

Doctoral Committee:
Associate Professor Carey N. Lumeng, Chair
Professor Cheong-Hee Chang
Professor Philip King
Associate Professor John Osterholzer
Assistant Professor Darleen Sandoval
Brian Francis Zamarron
[email protected]
ORCID ID: 0000-0001-6549-4230

© 2017 – Brian F. Zamarron
DEDICATION

To my mother, who wasn’t able to pursue the opportunities I’ve enjoyed
To my partner in mischief Alyson

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ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Carey Lumeng, for his support when I
approached him in 2012 about starting a series of weight loss experiments which would
requiring a series of 6-11 month long experimental setups. The results from those initial
experiments ended up being completely different from what we were expecting to find
and formed the basis of my entire doctoral research. I would like to also acknowledge the
members of my committee for their collective input towards my research design which
helped guide my projects at each stage.
I would also like to thank the members of the Lumeng laboratory for their help
over the years as well. Discussions about research, problems and, more often than not, pie
made my time here at Michigan quite unique. I’d be remiss to not specifically mention
the various undergraduate students which I have mentored over the years and became
capable and important members of the Lumeng laboratory. My time spent training and
mentoring these students was certainly rewarded back again with the help they’ve been
able to provide and I wish them the best of luck going forward.
Finally, I need to acknowledge the other members of the University of Michigan
Immunology Program. First the students – The discussions I’ve had with students has
covered a range of topics from simple research to existential life crises about life after
graduation. I’ll miss those I’ve grown close to, and I lament not having the time to
become better acquainted with others. I wish us all the best of luck regardless of our
future paths. Second is Bethany Moore and Zarinah Aquil – Beth has become a fantastic
strength for the Immunology program and I know the students appreciate her ridiculous
efforts made to help support the community. The same could be easily said of Zarinah,
whose job I’m sure feels about as easy as squeezing blood from a turnip more often than
not. Thank you both.
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TABLE OF CONTENTS
DEDICATION ........................................................................................................ ii
ACKNOWLEDGEMENTS ................................................................................... iii
LIST OF FIGURES ............................................................................................... vi
LIST OF TABLES ................................................................................................. ix
ABSTRACT ............................................................................................................ x
Chapter 1 – Introduction ......................................................................................... 1
Defining Obesity ................................................................................................. 1
Epidemiology of Obesity .................................................................................... 3
Comorbidities Associated with Obesity .............................................................. 4
Murine Models of Obesity .................................................................................. 6
Molecular Drivers of Obesity-Associated Insulin Resistance and Inflammation 9
Leukocyte Mediators of Obesity-Associated Inflammation ............................. 13
Scope of the Dissertation................................................................................... 19
Chapter 2 – Adipose Tissue Dendritic Cells are Independent Contributors to
Obesity-Induced Inflammation and Insulin Resistance ........................................ 24
Abstract ............................................................................................................. 24
Introduction ....................................................................................................... 25
Materials and Methods ...................................................................................... 27
Results ............................................................................................................... 32
Discussion ......................................................................................................... 45
Chapter 3 – Macrophage Proliferation Sustains Adipose Tissue Inflammation in
Formerly Obese Mice ........................................................................................... 51
Abstract ............................................................................................................. 51
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Introduction ....................................................................................................... 52
Materials and Methods ...................................................................................... 53
Results ............................................................................................................... 58
Discussion ......................................................................................................... 76
Chapter 4 – Weight Regain in Formerly Obese Mice Hastens the Development of
Hepatic Steatosis and Reveals Impaired Adipose Tissue Function ...................... 81
Abstract ............................................................................................................. 81
Introduction ....................................................................................................... 81
Materials and Methods ...................................................................................... 83
Results ............................................................................................................... 86
Discussion ....................................................................................................... 103
Chapter 5 – Conclusions and Future Directions ................................................. 109
Summary ......................................................................................................... 109
Objectives, Major Findings, and Implications for Chapter 2 .......................... 109
Objectives, Major Findings, and Implications for Chapter 3 .......................... 110
Objectives, Major Findings, and Implications for Chapter 4 .......................... 118
Final Thoughts................................................................................................. 124
REFERENCES ................................................................................................... 127

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LIST OF FIGURES

Figure 1-1 – Inhibitory effects of inflammation on insulin signaling ................... 11
Figure 1-2 – Cartoon depiction of leukocyte population changes that occur with
obesity and the resulting inflammatory signaling cascade that promotes insulin
resistance ............................................................................................................... 15
Figure 1-3 – Early progressive accumulation and activation of epididymal adipose
tissue T cells in diet-induced obese C57BL/6J mice ............................................ 17
Figure 1-4 – Polarization of pro-inflammatory CD11c+ macrophages in adipose
tissue during obesity ............................................................................................. 19
Figure 1-5 – Features normally associated with obesity persist in adipose tissue
despite weight loss ................................................................................................ 22
Figure 1-6 – Cartoon model depicting predicted metabolic perturbations after
HFD re-challenge .................................................................................................. 23
Figure 2-1 – CD64 is a specific ATM marker in lean and obese mice ................. 31
Figure 2-2 – Gene expression profiling of ATMs and ATDCs ............................ 34
Figure 2-3 – Myeloid DCs predominate in adipose tissue .................................... 36
Figure 2-4 – Time course of ATM and ATDC accumulation in adipose tissue with
HFD-induced obesity ............................................................................................ 37
Figure 2-5 – CCR2 is required for obesity-induced CD11c+ ATM and ATDC
migration into adipose tissue ................................................................................ 38
Figure 2-6 – CCR7 is required for ATDC accumulation during diet-induced
obesity ................................................................................................................... 41
Figure 2-7 – CCR7-deficient mice are protected from HFD-induced insulin
resistance and adipose tissue inflammation .......................................................... 42
Figure 2-8 – CD64– CD11c+ ATDCs are enriched in subcutaneous adipose tissue
from obese humans ............................................................................................... 44
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Figure 3-1 – Glucose tolerance normalization but persistently elevated insulin
with weight loss .................................................................................................... 57
Figure 3-2 – Epididymal adipose tissue maintains features associated with obesity
despite weight loss ................................................................................................ 59
Figure 3-3 – Maintenance of inflammatory CD11c+ adipose macrophages despite
weight loss ............................................................................................................ 64
Figure 3-4 – Obesity-induced effects can persist in adipose as long as six months
after HFD removal ................................................................................................ 67
Figure 3-5 – Inguinal adipose tissue and liver changes with weight loss ............. 68
Figure 3-6 – Adipose tissue T cell activation with weight loss ............................ 69
Figure 3-7 – T cells are not required for CD11c+ macrophage accumulation but
may control inflammatory activation state ........................................................... 70
Figure 3-8 – Rag1–/– mice show altered eWAT gene and protein expression during
obesity and weight loss similar to WT mice ......................................................... 74
Figure 3-9 – Macrophages are maintained through increased proliferation and
reduced apoptosis .................................................................................................. 75
Figure 3-10 – Adipose tissue dendritic cells have a less-inflammatory profile than
ATMs during obesity and weight loss .................................................................. 76
Figure 4-1 – Weight loss and HFD re-challenge model ....................................... 88
Figure 4-2 – HFD re-challenge increases epididymal adipose tissue crown-like
structures and reduces expression of proteins essential for mature adipocyte
development and function ..................................................................................... 90
Figure 4-3 – Inguinal adipose tissue shows resistance to derangement with HFD
re-challenge ........................................................................................................... 91
Figure 4-4 – Increased liver steatosis and signs of liver damage with HFD re-
challenge ............................................................................................................... 93
Figure 4-5 – Liver lipid metabolism and lipogenesis gene expression is not
significantly different between ST HFD and RC HFD mice groups .................... 94
Figure 4-6 – HFD re-challenge after 24 weeks weight loss cycle reveals similar
defects in eWAT expansion capacity .................................................................... 97
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Figure 4-7 – Increased liver triglycerides and steatosis after HFD re-challenge of
extended weight loss cycle mice ........................................................................... 98
Figure 4-8 – Increased CD11c+ ATM accumulation and inflammation with HFD
re-challenge ........................................................................................................... 99
Figure 4-9 – eWAT of formerly obese mice maintains elevated numbers
preadipocytes ...................................................................................................... 100
Figure 4-10 – Conditioned media from eWAT explants of formerly obese mice
inhibits adipocyte differentiation and reduces lipogenesis ................................. 101
Figure 5-1 – Development of features normally associated with chronically-obese
adipose tissue in the eWAT of formerly obese mice .......................................... 111
Figure 5-2 – Weight gained before weight loss influences maintenance of obesity-
associated features in eWAT during weight loss. ............................................... 115
Figure 5-3 – Model showing failure of eWAT preadipocytes to properly respond
to differentiation signals upon HFD re-challenge of formerly obese mice which
leads to faster development of hepatic steatosis ................................................. 122

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LIST OF TABLES

Table 1 – Classification of obesity and comorbidity risk in adults. ....................... 2
Table 2 – Body fat percent in C57BL/6 male mice increases with body weight.... 8
Table 3 – Relative leukocyte population changes in murine epididymal adipose
tissue with obesity. ................................................................................................ 14
Table 4 – Antibodies used for flow cytometry this chapter .................................. 49
Table 5 – Sequences for RT-PCR primers used in this chapter ............................ 50
Table 6 – Sequences for RT-PCR primers used in this chapter. ........................... 80
Table 7 – Sequences for RT-PCR primers used in this chapter .......................... 108

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