2 L - JScholarship - Johns Hopkins University

THE ROLE OF INTRAMYOCELLULAR FATTY ACIDS ON THE ETIOLOGY OF THE INSULIN
RESISTANCE OF OBESITY






by
Chryssanthi Stylianopoulos








A dissertation submitted to the Johns Hopkins University in conformity with
the requirements for the degree of Doctor of Philosophy


Baltimore, Maryland
December 2005










( Chryssanthi Stylianopoulos 2005
All rights reserved
ABSTRACT
The intracellular concentration of fatty acids in insulin-sensitive
cells is purported to be a key factor in the development of insulin
resistance. It is hypothesized that the intramyocellular lipid (IMCL)
concentration is correlated with the degree of insulin resistance (IR), and
that a reduction in IMCL will have a more significant effect on IR than a
reduction in body adipose tissue stores. This study assessed the effects of
weight loss through dietary intervention on the IMCL and IR on a group of
obese adults and explored the correlations between IR, IMCL, body mass
index (BMI), serum triglycerides (TG), free fatty acids, and total body fat
of obese adults with an otherwise similar group of lean individuals.
Baseline tests were performed in lean and obese, including a 2 hour Oral
Glucose Tolerance Test (OGTT), body composition measurement by Dual Energy
X-Ray Absorptiometry (DEXA), and IMCL determination in the tibialis
anterioris (TA) and soleus (SOL) muscles by proton magnetic resonance
spectroscopy. The obese underwent insulin sensitivity assessment by the
euglycemic-hyperinsulinemic clamp. Furthermore, they were instructed to
follow a hypocaloric diet, and were subsequently re-evaluated after a
weight loss of 8-10% of total body weight.
Our results indicate that the obese group had significantly higher IMCL
levels in the SOL muscle than the lean group. In both groups, there was a
significant positive correlation of IR, assessed by Homeostasis Model
Assessment Index (HOMA), with IMCL in the SOL muscle but not in the TA.
IMCL in the SOL was an important predictor of IR by HOMA, after controlling
for age, BMI, and TG. Weight loss resulted in a significant decrease in IR
and IMCL in the TA, but not in the SOL muscle. There was no correlation
between changes in IMCL in TA myocytes and in IR. Our data documented that
reduction in IMCL stores might play an important role in insulin signaling.


Thesis Advisor: Dr. Benjamin Caballero
Thesis Committee: Drs. Christopher Saudek, Benjamin Caballero, Alena
Horska, Margarita Treuth, Eliseo Guallar

ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Benjamin Caballero, who provided
me with the support and guidance for the successful completion of this
project.
I would like to thank Dr. Christopher Saudek for financial support
through the National Research Service Award Research Training Grant. I
would also like to thank the Johns Hopkins University School of Medicine
General Clinical Research Center (award M01-RR00052, from the National
Center for Research Resources/National Institutes of Health) and the United
Stated Department of Agriculture (award 58-1235-2-060) for financial
support of this project.
I would like to thank Dr. Alena Horska for assistance on the radiology
component of this project. I would also like to thank Terri Brawner and
Kathleen Kahl for technical support with the magnetic resonance scanner.
I would like to thank Dr. Lawrence Cheskin and Amy Mitchell for their
contribution on the weight management part of this project.
I would like to thank Dr. Todd Brown and Dr. Alice Ryan for their help
with the insulin clamp procedure.
I would also like to thank Mary Missouri, Suzanne Dunphy and all the
nurses and staff at the General Clinical Research Center for their
invaluable collaboration.
Most of all, I would like to thank my family and friends for their
continuous love and support throughout this journey.
TABLE OF CONTENTS
1 INTRODUCTION 1
2 LITERATURE REVIEW 4
2.1 Insulin Actions: Overview 4
2.2 Insulin receptor 5
2.3 Insulin Resistance, Insulin Insensitivity and Insulin Unresponsiveness
6
2.4 Insulin and Leptin 7
2.5 Insulin and Adiponectin 9
2.6 From Insulin Resistance to Type 2 Diabetes 10
2.7 Type 2 Diabetes 10
2.8 Diagnosis of Diabetes 11
2.9 Genetic Contributions to Type 2 Diabetes 12
2.10 Obesity-Induced Type 2 Diabetes 14
2.10.1 Receptor Events 14
2.10.2 Post-receptor Events 16
2.11 Adipose Tissue Deposition in Human Skeletal Muscle and Liver 16
2.11.1 Quantification Methods 16
2.11.2 Proton Magnetic Resonance Spectroscopy 17
2.11.3 Mechanism of Muscle Lipid Accumulation 20
2.11.4 Fatty Liver Disease 22
2.11.5 Lipid Accumulation and Insulin Resistance 22
2.11.6 Lipid Accumulation and Exercise 28
2.11.7 Lipid Accumulation and Adiponectin 29
3 HYPOTHESES AND OBJECTIVES 31
3.1 Hypotheses 31
3.2 Objectives 31
4 MATERIALS AND METHODS 33
4.1 Study Design 33
4.2 Sample Size 34
4.3 Subjects 35
4.4 Baseline Measurements 36
4.4.1 Initial Screening 36
4.4.2 Proton Nuclear Magnetic Resonance Spectroscopy 38
4.4.3 Euglycemic-Hyperinsulinemic Clamp Procedure 41
4.5 Dietary Intervention 42
4.6 Follow-up measurements 44
4.7 Laboratory Procedures 45
4.8 Insulin Sensitivity Assessments 46
4.9 Proton Magnetic Resonance Spectroscopy Data Processing 48
4.10 Statistical Analysis 50
5 RESULTS 52
5.1 Subject Characteristics 52
5.2 Baseline Comparisons 52
5.2.1 Anthropometric Data and Fasting Blood Tests 52
5.2.2 Intracellular Lipid Accumulation 55
5.2.3 Euglycemic-Hyperinsulinemic Clamp 62
5.2.4 Correlations 64
5.2.5 Insulin Resistance Predictors 68
5.3 Follow up 69
5.3.1 Anthropometric Data and Fasting Blood Tests 70
5.3.2 Intracellular Lipid Accumulation 72
5.3.3 Losses to Follow up 76
5.3.4 Correlations 78
6 DISCUSSION 80
6.1 Intracellular Lipid Concentration is Higher in Obese Compared to Lean
80
6.2 Intracellular Lipid Levels are Positively Correlated with Insulin
Resistance and Glucose Intolerance 80
6.3 Weight Loss Reduces Intracellular Lipid 82
6.4 Other Factors Affecting Intramyocellular Lipid Concentration 84
6.4.1 Muscle Fiber Type 84
6.4.2 Physical Activity 86
6.4.3 Adiponectin 87
6.5 Proton Magnetic Resonance Spectroscopy 88
6.6 Measures of Insulin Resistance 89
6.7 Suggestions for Future Research 90
6.8 Summary and Conclusions 92
7 REFERENCES 94
8 CURRICULUM VITAE 117
LIST OF TABLES
1 Study
methodology.................................................................
.............. 45
2 Spectral
parameters..................................................................
............. 49
3 Baseline anthropometric characteristics of lean and
obese.................................. 53
4 Baseline plasma lipid levels of lean and
obese................................................ 54
5 Baseline plasma hormones levels, insulin sensitivity and glucose
metabolism indices
of lean and
obese........................................................................
......... 55
6 Baseline IMCL, EMCL, and IHL by proton magnetic resonance
spectroscopy of
lean and
obese........................................................................
............ 56
7 Baseline euglycemic-hyperinsulinemic clamp data of obese
subjects....................... 63
8 Anthropometric characteristics of obese subjects before and
after weight loss............ 70
9 Plasma lipid levels of obese subjects before and after weight
loss.......................... 71
10 Plasma hormones levels, insulin sensitivity and glucose metabolism of
obese subjects
before and after weight
loss.....................................................................
72
11 IMCL, EMCL, and IHL by proton magnetic resonance spectroscopy of
obese
subjects before and after weight
loss............................................................ 73
12 Anthropometric characteristics of obese subjects that completed
the weight loss
compared to those who dropped
out.......................................................... 76
13 Plasma metabolite concentrations of obese subjects that
completed the weight loss
compared to those who dropped
out.......................................................... 77
14 IMCL, EMCL, and IHL by proton magnetic resonance spectroscopy
of obese subjects
that completed the weight loss compared to those who dropped
out..................... 78
LIST OF FIGURES
1 Mobilization of GLUT4 from intracellular stores to cell
surface............................. 6
2 Types of insulin
resistance..................................................................
...... 7
3 Proton spectrum of human soleus
muscle................................................