Table Of ContentTHE ACUTE IMPACT OF A SINGLE DOSE OF
RESVERATROL ON INSULIN SENSITIVITY, WHOLE BODY FAT
OXIDATION, AND INTRACELLULAR SIGNALING IN SKELETAL
MUSCLE AND ADIPOSE TISSUE IN OVERWEIGHT AND OBESE
MEN
by
Cameron B. Williams
A thesis submitted to the School of Kinesiology and Health Studies
In conformity with the requirements for
the degree of Master of Science.
Queen’s University
Kingston, Ontario, Canada
(June, 2013)
Copyright © Cameron B. Williams, 2013
Abstract
Resveratrol (RSV) is a natural compound that improves mitochondrial function and
metabolic health in animal models. Thus far, RSV’s effects on metabolic outcomes in humans are
controversial, and RSV’s acute mechanism has not yet been confirmed in vivo. This study aimed
to evaluate the effect of an acute dose of RSV on insulin sensitivity and fatty acid oxidation, and
to determine RSV’s mechanism of action in human skeletal muscle and adipose tissue.
Overweight males (n=8; BMI, 30.5±3.6; VO peak, 34.0±7.3 ml/kg) reported to the lab on 2
2
occasions and were provided a breakfast supplemented with 0.3g of RSV or a placebo pill.
Experiments were performed in random order using a double blind crossover design. Gas
exchange measures, blood samples, and skeletal muscle and adipose tissue biopsies were obtained
before and 2 hours after the supplement meal. RSV acutely improved insulin sensitivity, but had
no effect on fatty acid oxidation. Additionally, RSV supplementation had no effect on the
intracellular signaling of key proteins proposed to mediate its effects in skeletal muscle and
adipose tissue. Taken together, these results suggest a single dose of RSV can acutely enhance
insulin sensitivity, but its mechanism of action is not conserved across species, and its
intracellular signaling pathway is different in humans than previously thought. Due to its insulin
sensitizing effect, RSV retains its clinical value, but further research is required to determine its
most useful application for human metabolic health.
ii
Co-Authorship
This thesis presents the work of Cameron Williams in collaboration with Dr. Brendon Gurd.
The impact of a single dose of resveratrol on insulin sensitivity, whole body fat oxidation, and
intracellular signaling in skeletal muscle and adipose tissue in overweight and obese men is
presented according to the guidelines for the Journal of Physiology. Cameron Williams was
responsible for reviewing relevant literature to identify the research question, conducting the
intervention, performing the data analyses, and drafting the manuscript. All aspects were a
collaborative effort between Cameron Williams and Dr. Brendon Gurd. In addition, Dr. Craig
Simpson provided medical supervision and contributed to study design. Finally Brittany Edgett
was instrumental in blood collection.
Chapter 2 is a reproduction of Skeletal muscle SIRT1 and the genetics of metabolic health:
therapeutic activation by pharmaceuticals and exercise originally published August, 2012 in The
Application of Clinical Genetics. Cameron Williams was responsible for reviewing the relevant
literature and drafting the manuscript. The paper was co-authored by Dr. Brendon Gurd. It has
been reproduced herein with the express permission of both the authors (C.B. Williams and B.J.
Gurd) and the approval of the editorial board of The Application of Clinical Genetics and Dove
Medical Press Ltd. Please see Appendix D for written permission.
iii
Acknowledgements
I would like to begin by thanking my supervisor, Dr. Brendon Gurd. Choosing to pursue
a Master’s degree under his tutelage was an important and rewarding academic decision. He has
invested a lot of time and money into me, and continually provided the support and guidance I
have needed to maintain forward progress in my academic and scientific career. So thank you
Brendon, it means a lot.
I would also like to thank my lab mates that have helped, supported, and enhanced every
aspect of my grad school experience. I couldn’t ask for a better group of people to work with. A
lot of laughs and a lot of memories! Brit, Tina, Trish, Marysa, Jasmin, Colin, Mel, and Jason.
Thanks guys!
I would also like to thank Dr. Michael Tschakovsky for his continued mentorship from
my undergrad into my graduate studies. It was his offer in my 4th year that first introduced me to
research and was likely the catalyst for my accepting a graduate school position. Thanks Dr. T.
Last, but definitely not least, I would like to thank my parents for all the love, help, and
support they have given me through my entire academic career and more importantly my life.
I’ve got a couple years left so hopefully you aren’t ready to throw in the towel just yet!
I have truly enjoyed my time in the Gurd Lab and won’t soon forget the past two years.
Thank you to everyone who was part of that!
iv
Table of Contents
Abstract ............................................................................................................................................ ii
Co-Authorship ................................................................................................................................. iii
Acknowledgements ......................................................................................................................... iv
List of Figures ............................................................................................................................... viii
List of Tables ................................................................................................................................... ix
List of Abbreviations ........................................................................................................................ x
Chapter 1 Introduction ..................................................................................................................... 1
1.1 General Introduction .............................................................................................................. 1
1.2 Metabolic Function: Importance of Mitochondria ................................................................. 2
1.3 Resveratrol: A Promising Candidate As An Exercise Mimetic ............................................. 3
1.4 Thesis Objectives and Experimental Approach ..................................................................... 4
1.5 Thesis Organization ............................................................................................................... 5
1.6 References .............................................................................................................................. 5
Chapter 2 Literature Review: SIRT1 and Metabolic Health ............................................................ 9
2.1 Abstract .................................................................................................................................. 9
2.2 Introduction .......................................................................................................................... 10
2.3 Skeletal Muscle Mitochondria: Mechanisms of Metabolic Disease .................................... 12
2.4 Genetic Control of Skeletal Muscle Mitochondrial Content: The SIRT1/PGC-1α Axis ..... 16
2.4.1 Physiological Control of SIRT1 Activity ...................................................................... 18
2.4.2 Pharmacological Activation of SIRT1 .......................................................................... 20
2.5 Targeting SIRT1 activation as therapeutic intervention ...................................................... 21
2.5.1 Health Benefits Associated with Pharmaceutical Activation of SIRT1 ........................ 21
2.5.2 Health Benefits of SIRT1-PGC1α Axis Activation During Exercise ........................... 24
2.6 Conclusion and Future Direction ......................................................................................... 26
2.7 References ............................................................................................................................ 27
Chapter 3 Literature Review: Resveratrol ...................................................................................... 39
3.1 Overview .............................................................................................................................. 39
3.2 Resveratrol ........................................................................................................................... 39
3.2.1 Resveratrol’s Role in Metabolic Health ........................................................................ 40
3.2.2 Insulin Sensitivity .......................................................................................................... 41
v
3.2.3 Fatty Acid Oxidation ..................................................................................................... 42
3.2.4 Further Investigation Required ..................................................................................... 43
3.3 Resveratrol’s Mechanism of Action in Skeletal Muscle ...................................................... 43
3.3.1 Calcium/Calmodulin Dependent Pathway .................................................................... 44
3.3.2 SIRT1 Dependent Pathway ........................................................................................... 44
3.3.3 Resveratrol’s Mechanism in Humans ........................................................................... 46
3.3.4 Insulin Sensitivity .......................................................................................................... 47
3.3.5 Fatty Acid Oxidation ..................................................................................................... 48
3.4 Adipose Tissue and Metabolic Health ................................................................................. 49
3.5 Resveratrol’s Mechanism of Action in Adipose Tissue ....................................................... 51
3.5.1 Adipose Tissue Mitochondrial Biogenesis .................................................................... 52
3.6 Thesis Purpose ..................................................................................................................... 54
3.7 Thesis Hypotheses ................................................................................................................ 54
3.8 References ............................................................................................................................ 55
Chapter 4 A Single Dose of Resveratrol Acutely Improves Insulin Sensitivity, But Has No Effect
On Whole Body Fat Oxidation or SIRT1 Activation in Skeletal Muscle and Adipose Tissue in
Overweight and Obese Men ........................................................................................................... 60
4.1 Abstract ................................................................................................................................ 60
4.2 Introduction .......................................................................................................................... 61
4.3 Methods ................................................................................................................................ 64
4.3.1 Participants .................................................................................................................... 64
4.3.2 Experimental Protocol ................................................................................................... 65
4.3.3 Baseline Testing ............................................................................................................ 65
4.3.4 Supplement Intervention Protocol ................................................................................. 65
4.3.5 Physiological Measurements ......................................................................................... 67
4.3.6 Fasting Glucose, Insulin, HOMA-IR and Estimates of Substrate Utilization ............... 67
4.3.7 Western Blot Analysis ................................................................................................... 68
4.3.8 Statistical Analysis ........................................................................................................ 69
4.4 Results .................................................................................................................................. 72
4.4.1 HOMA-IR, Insulin and Glucose ................................................................................... 72
4.4.2 Fatty Acid Oxidation ..................................................................................................... 72
4.4.3 Skeletal Muscle Signaling ............................................................................................. 73
vi
4.4.4 Adipose Tissue Signaling .............................................................................................. 73
4.5 Discussion ............................................................................................................................ 78
4.5.1 Resveratrol and Insulin Sensitivity ............................................................................... 78
4.5.2 Whole Body Fatty Acid Oxidation ............................................................................... 80
4.5.3 Resveratrol and SIRT1 activation in skeletal muscle ................................................... 81
4.5.4 Cellular signaling in Adipose Tissue ............................................................................ 83
4.6 Limitations ........................................................................................................................... 84
4.7 Summary .............................................................................................................................. 85
4.8 References ............................................................................................................................ 86
Chapter 5 General Discussion ........................................................................................................ 93
5.1 Summary of Key Findings ................................................................................................... 93
5.2 Study Strengths .................................................................................................................... 93
5.3 Study Implications ............................................................................................................... 94
5.4 Study Limitations ................................................................................................................. 95
5.5 Future Research .................................................................................................................... 96
5.6 M.Sc. Research Experience .................................................................................................. 97
5.7 Conclusions .......................................................................................................................... 98
5.8 References ............................................................................................................................ 98
Appendix A Research Ethics Board - Letter of Informed Consent ............................................. 100
Appendix B Muscle Biopsy Information Sheet ........................................................................... 110
Appendix C Research Ethics Board Approval ............................................................................. 115
Appendix D Dove Medical Press Permission to Reproduce Publication .................................... 118
vii
List of Figures
Figure 1. Pathways involved in the SIRT1-PGC-1α axis. ............................................................. 15
Figure 2. Proposed mechanism of resveratrol action in skeletal muscle. ...................................... 46
Figure 3. Proposed mechanism of resveratrol action in adipose tissue. ......................................... 53
Figure 4. Experimental Visit Timing. ............................................................................................ 67
Figure 5. Impact of an acute dose of resveratrol (RSV) on 2 hour insulin sensitivity. .................. 74
Figure 6. Whole body fat oxidation is unchanged by resveratrol (RSV). ...................................... 75
Figure 7. Resveratrol alters p38 MAPK phosphorylation but does not alter other intramuscular
signaling pathways. ........................................................................................................................ 76
Figure 8. Adipose tissue signaling is unaltered by resveratrol. ...................................................... 77
viii
List of Tables
Table 1. Subject characteristic summary. ...................................................................................... 71
ix
List of Abbreviations
ACC – acetyl-CoA carboxylase
AICAR – 5-aminioimidazole-4-carboxamid ribonucleotide
AMP – adenosine monophosphate
AMPK – AMP-activated protein kinase
ANOVA – analysis of variance
AROS – active regulator of SIRT1
ATP – adenosine triphosphate
BMI – body mass index
cAMP – cyclic 3’5’-adenosine monophosphate
CamKII – calcium/calmodulin-dependent protein kinase II
CamKKβ - calcium/calmodulin-dependent protein kinase kinase-β
CHO – carbohydrate
cm – centimeter
CPT-1 – carnitine palmitoyltransferase
CV – confidence variable
DAG – diacylglycerol
DBC-1 – deleted in breast cancer-1
DNA – deoxyribonucleic acid
EDTA – ethylene diamine tetra-acetic acid
EGTA – ethylene glycol tera-acetic acid
ELISA – enzyme-linked immunoabsorbent assay
Epac1 – exchange protein activated by cAMP-1
x
Description:Resveratrol (RSV) is a natural compound that improves mitochondrial function
and metabolic health in animal models. 3.3 Resveratrol's Mechanism of
Action in Skeletal Muscle . metabolism in association with insulin resistance,
obesity, and weight loss. groups of US middle-aged and older-aged wom
