pharmacological and cold-induced alterations in glucose and fat utilization

285 Pages·2017·6.5 MB·English

PHARMACOLOGICAL AND COLD-INDUCED ALTERATIONS IN
GLUCOSE AND FAT UTILIZATION: IMPLICATIONS FOR TISSUE-
SPECIFIC AND WHOLE-BODY ENERGY METABOLISM

DIANE MIEKO SEPA-KISHI

A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE
STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR
THE DEGREE OF

DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN KINESIOLOGY AND HEALTH SCIENCE
YORK UNIVERSITY
TORONTO, ONTARIO
NOVEMBER 2017
©Diane Mieko Sepa-Kishi, 2017
Abstract
This dissertation presents two possible approaches to enhancing glucose
utilization, improving insulin sensitivity and reducing fat mass that could have
therapeutic value for the treatment of obesity and type 2 diabetes (T2D). The results of
my first study showed that suppressing the import of long-chain fatty acids into the
mitochondria improved glucose homeostasis while also reducing fat mass. A reduction
in lipogenesis in the white adipose tissue (WAT) explained the reduction in fat mass as
lipolysis was also reduced. Subsequent studies examined the effects that activation of
cold-induced thermogenesis would have on whole-body and tissue-specific metabolism.
I confirmed that cold exposure increases UCP1 content (induces browning) in the
subcutaneous (Sc) inguinal (Ing) WAT, but it did not enhance substrate oxidation in this
fat depot. Instead, cold acclimation activated a futile cycle of lipolysis and lipogenesis
that contributed to increasing energy expenditure within this fat depot. This futile cycle
was not activated in the epididymal (Epid) fat, confirming that cold induced a fat depot-
specific browning adaptive response. I then studied the hormone fibroblast growth factor
21 (FGF21) as a factor that could explain this depot-specific difference. FGF21 content
and secretion was enhanced in the Sc Ing WAT, but not the Epid WAT depot. The
downstream signaling pathway was also only activated in the Sc Ing WAT depot,
suggesting that FGF21 could be involved in the depot-specific browning that I observed.
Additional studies showed that muscles rich in both type I and type II fibers enhanced
their FA oxidation following cold exposure. However it was only muscles rich in type I
fibers that enhanced expression of the glucose transporter Glut4 while also reducing
phosphorylation of glycogen synthase, leading to an increase in glycogen synthesis rate
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and glycogen content after cold acclimation. Liver glycogen content was reduced
following cold acclimation, while gluconeogenesis was enhanced in this organ. Despite
a cold-induced increase in food intake and endogenous glucose production, and
reduction in plasma insulin, circulating glucose remained unchanged. These results
show that activation of cold-induced thermogenesis can enhance insulin sensitivity and
could be a viable alternative treatment for diseases such as obesity and T2D.

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Acknowledgements
Dr. Rolando Ceddia: None of this would have been possible without your guidance and
support. These past 4 years have simultaneously been the toughest and most
rewarding of my entire life. Since day 1 you’ve had my back and with you as my mentor
I’ve managed to accomplish so much more than I ever thought I would during my PhD.
Most importantly, our relationship has been one of collaboration and friendship, and I
am grateful for that. A thank you doesn’t even begin to express my gratitude. A large
can of mixed nuts might…

Ceddia Lab members: Michelle, Abinas and Arta, we were the original 4. Thank you for
making me laugh and making the long days bearable. Michelle especially, you will
always be the best lab partner ever. Pauline, our friendship and collaborations have
been such a blessing. Thank you for all types of science discussions and for taking care
of me in Brazil. Ishvinder, thanks for being a buddy and accompanying me to
conferences and presentations so I could be social my last year here. Dr. Ricardo
Pinho, obrigada pela confiança e pela oportunidade de viajar e trabalhar no Brasil. It
was an experience I will never forget! George, you are a PCR master and have taught
me so much. There was never a dull day with you in the lab! And to every other
volunteer and undergraduate student who graced the Ceddia Lab over the last 4 years-
there are too many to list but thank you for every task you completed, big or small, that
helped me complete my work.

My supervisory committee: Your guidance and support are much appreciated. Thank
you for being thoughtful, critical, patient and encouraging at all the right times.

My family and friends: Mom, Dad, my brother Matt, my grandparents, friends,
teammates and the countless cousins and Aunts and Uncles I have, thank you for
always asking how research was going, even if you had no idea what it entailed. Thank
you for the laughter, food, competition and company that provided such a balance to my
life as a grad student and helped to keep me sane.

Peit: Nobody quite understands the stress of a grad student quite like their spouse.
Thank you for being a sounding board for all my frustrations and for making me laugh or
look at pictures of puppies when I really needed to. Thanks for your patience and
understanding for every weekend or late night spent in the lab, for your pride in my
work, and for loving me no matter what. And thank you for working so hard so that I
never ever had to worry about paying bills while trying to complete my research.

The Elia Family: Thank you for your generous contribution to York University in the form
of the Elia Scholars program that allowed me to invest all of my time and energy into my
research. I’m proud to be part of such a prestigious group of individuals.

Lastly, thanks be to God, without whom none of this would be possible.
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Table of Contents
Abstract ........................................................................................................................ii
Acknowledgements .....................................................................................................iv
List of Figures .............................................................................................................. x
List of Tables ............................................................................................................. xiv
List of Abbreviations ...................................................................................................xv
CHAPTER 1: Introduction ............................................................................................... 1
CHAPTER 2: Literature Review ...................................................................................... 4
2.1. White Adipose Tissue (WAT) ................................................................................ 4
2.1.1. Structure and depot-specific differences ........................................................ 4
2.1.2. Function of the WAT and contribution to energy homeostasis ....................... 5
2.1.2.1. Regulation of fatty acid synthesis and TAG formation ................................. 6
2.1.2.2. Regulation of lipolysis ................................................................................ 10
2.2. Brown Adipose Tissue (BAT) .............................................................................. 16
2.2.1. Structure and function .................................................................................. 16
2.2.2. Development of BAT .................................................................................... 17
2.2.3. Activation of the BAT .................................................................................... 20
2.2.4. Browning of the WAT ................................................................................... 22
2.3. Skeletal muscle .................................................................................................. 28
2.3.1. Structure and excitation-contraction coupling ............................................... 28
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2.3.2. ATP production in skeletal muscle ............................................................... 29
2.3.3. Skeletal muscle fiber type characterization .................................................. 32
2.3.4. Skeletal muscle contribution to energy homeostasis .................................... 34
2.3.5. Skeletal muscle adaptation to cold ............................................................... 38
2.4. Liver .................................................................................................................... 42
2.4.1. Glucose metabolism in the liver ................................................................... 43
2.4.1.1. Glycogen synthesis and breakdown .......................................................... 43
2.4.1.2. Lipogenesis ............................................................................................... 45
2.4.1.3. Gluconeogenesis....................................................................................... 48
2.4.2. Fatty acid metabolism in the liver ................................................................. 51
2.4.3. Liver adaptation to cold ................................................................................ 52
2.5. Obesity and T2D: Pathophysiology and Treatment ............................................ 53
2.5.1. Alterations in Energy Metabolism with Obesity and T2D .............................. 53
2.5.2. The Therapeutic Potential of BAT ................................................................ 57
2.5.3. Pharmacological inhibition of β-oxidation as a therapy for obesity and T2D 60
CHAPTER 3: Objectives and Hypotheses ..................................................................... 67
CHAPTER 4: Antilipolytic and antilipogenic effects of the CPT-1b inhibitor oxfenicine in
the white adipose tissue of rats ..................................................................................... 72
4.1. Abstract .............................................................................................................. 74
4.2. Introduction ......................................................................................................... 75
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4.3. Materials and methods ....................................................................................... 77
4.4. Results ................................................................................................................ 82
4.5. Discussion .......................................................................................................... 91
CHAPTER 5: Activation of a futile cycle rather than mitochondria uncoupling promotes
energy dissipation in cold-induced browning of the subcutaneous inguinal white adipose
tissue in rats .................................................................................................................. 99
5.1. Abstract ............................................................................................................ 101
5.2. Introduction ....................................................................................................... 102
5.3 Materials and Methods ...................................................................................... 105
5.4. Results .............................................................................................................. 110
5.5. Discussion ........................................................................................................ 119
CHAPTER 6: Cold-induced autocrine/paracrine effects of fibroblast growth factor 21:
Implications for depot-specific white adipose tissue browning in rats .......................... 124
6.1. Abstract ............................................................................................................ 126
6.2. Introduction ....................................................................................................... 127
6.3. Materials and Methods ..................................................................................... 130
6.4. Results .............................................................................................................. 133
6.5. Discussion ........................................................................................................ 141
CHAPTER 7: Cold acclimation causes fiber type-specific responses in glucose and fat
metabolism in rat skeletal muscles .............................................................................. 146
7.1. Abstract ............................................................................................................ 148
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7.2. Introduction ....................................................................................................... 149
7.3. Materials and Methods ..................................................................................... 152
7.4. Results .............................................................................................................. 156
7.5. Discussion ........................................................................................................ 164
CHAPTER 8: Cold acclimation reduces hepatic protein kinase B and AMP-activated
protein kinase phosphorylation and increases gluconeogenesis in rats ...................... 171
8.1. Abstract ............................................................................................................ 173
8.2. Introduction ....................................................................................................... 174
8.3. Materials and Methods ..................................................................................... 177
8.4. Results .............................................................................................................. 181
8.5. Discussion ........................................................................................................ 192
CHAPTER 9: Integrated Summary .............................................................................. 198
CHAPTER 10: Study Limitations ................................................................................. 204
CHAPTER 11: Future Directions ................................................................................. 206
References .............................................................................................................. 209
Appendix A: Supplementary Data ............................................................................ 235
Appendix B: Detailed Experimental Methods ........................................................... 243
B-1: Adipocyte Isolation ....................................................................................... 243
B-2: Complexation of Palmitate ............................................................................ 243
B-3: Determination of FFA using Wako HR Series NEFA-HR kit ......................... 244
B-4: Palmitate and Glucose oxidation .................................................................. 245
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B-5: Glycerol Determination as a Measure of Lipolysis ........................................ 246
B-6: Glucose or Glycerol Incorporation into Lipids ............................................... 246
B-7: Glycogen Synthesis ...................................................................................... 247
B-8: Glycogen Content ......................................................................................... 247
B-9: Real-Time Quantified Polymerase Chain Reaction (qPCR) .......................... 248
B-10: Western Blotting ......................................................................................... 250
B-11: ELISA kits for the determination of plasma insulin, glucagon, and FGF21 . 254
B-12: TAG Quantification using a Colorimetric Kit (BioVision Cat# K622-100) .... 257
Appendix C: Additional Contributions ...................................................................... 259
Appendix D: Published Work ................................................................................... 260

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List of Figures
Figure 2-1: Unilocular whilte adipocyte. .......................................................................... 5
Figure 2-2: Synthesis of G3P for TAG formation. ........................................................... 8
Figure 2-3: Schematic of the lipolytic signaling pathway. ............................................. 11
Figure 2-4: Multilocular brown adipocyte. ..................................................................... 17
Figure 2-5: Brown, beige and white adipocyte origin and development. ...................... 18
Figure 2-6: The FGF21 signaling pathway. .................................................................. 27
Figure 2-7: Schematic of glucose uptake into skeletal muscle. .................................... 35
Figure 2-8: AMPK signaling pathway. .......................................................................... 37
Figure 2-9: Regulation of SERCA by SLN. ................................................................... 40
Figure 2-10: Schematic of the lipogenesis pathway. .................................................... 46
Figure 2-11: Schematic of the gluconeogenic pathway in the liver. .............................. 50
Figure 2-12: Import of LCFA into the mitochondria for β-oxidation. .............................. 62
Figure 4-1: Oxfenicine does not affect VO or ambulatory activity, but reduces whole-
2
body fat oxidation and increases circulating non-esterified fatty acids (NEFAs). .......... 83
Figure 4-2: Oxfenicine normalizes fasting insulin and improves insulin sensitivity. ...... 85
Figure 4-3: Oxfenicine does not affect energy intake (B), but reduces body weight (A)
and Epid (C) and Sc Ing (D) fat pad mass. .................................................................... 86
Figure 4-4: Administration of oxfenicine reduces palmitate oxidation in epididymal (A)
and subcutaneous inguinal (B) adipocytes. ................................................................... 88
Figure 4-5: Glycerol release is reduced in subcutaneous inguinal (B) but not in
epididymal (A) adipocytes from LF- or HF-fed rats either injected with PBS or oxfenicine
(Ox, 150 mg/kg of BW). ................................................................................................. 88
x