Subclinical atherosclerosis in overweight Latino youth: influence of cardiometabolic risk factors

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SUBCLINICAL ATHEROSCLEROSIS IN OVERWEIGHT LATINO YOUTH:
INFLUENCE OF CARDIOMETABOLIC RISK FACTORS

by
Claudia M. Toledo-Corral
______________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL OF THE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PREVENTIVE MEDICINE)

December 2010

Copyright 2010 Claudia M. Toledo-Corral

ii
ACKNOWLEDGEMENTS

My journey on the academic road has been long and arduous. My love for the
biological sciences began at a very early age when I indulged in the anatomy and health
chapters of my childhood encyclopedia. In the fourth grade, I wanted to understand why
cancer existed and I fantasized about discovering a cure. In subsequent years, I
discovered the road to becoming a successful scientist would be difficult and I would
need much guidance. I now have many people to thank for successfully reaching my
goal.
I had many meaningfully mentoring relationships since my undergraduate years.
Dr. Pamela Viele and Dr. Mariaelena Lara were the first advisors who avidly encouraged
my academic pursuits. At CSUN, Dr. Lisa Banner and Dr. Maria Elena Zavala nurtured
my potential while providing constructive criticism. At USC, my first advisor, Dr. Martha
Cruz, always reminded me that no matter how well I did I would always have to work
harder. Dr. Jaimie Davis who has helped me tremendously as an unspoken committee
member and as a friend. I’d like to thank my dissertation committee members. Dr.
Michael Goran has shown an unwavering support and guidance, for which I am
extremely grateful. Dr. Marc Weigensberg who has always made me feel like a true
colleague and has patiently helped me progress with my critical thinking skills. Dr.
Howard Hodis, Dr. Wendy Mack, and Dr. Richard Watanabe, thank you for offering your
input during critical times. Your guidance will prove to be invaluable during the early
stages of my academic career.
Outside of the academic world, I have also been blessed with strong social
support networks. First and foremost, I would not have reached my goal if it were not for
the love and patience of my parents, Maria Lozada Toledo and Juan Antonio Toledo.
They nurtured my academic potential and continually supported me without hesitation.

iii
My sister, April Toledo, who has been undeniably been my best confidant and girl friend.
My husband, Pete Corral, who has been my foundation and being of logic; without you, I
am not whole. I love you and thank you for all your loving support. Thank you to my
dearest friends that I met during tumultuous times at UCLA: Dr. Marla Almazan, Jennifer
Madrigal, and Dr. Judith Sanchez. We have shared so much over the past 13 years and
I am so thankful our friendships have stood the test of time; a true testament to the
strength of our relationships. Thank you to David Dain, with whom I share my longest
standing friendship and as a consequence witnessed my commitment to academia since
the age of 12. My other dear friends, Dr. Raquel Martinez, Dr. Emily Ventura and Monica
Zepeda Luarca; with you I shared the road of academic challenge and along the way we
grew very close and I cannot imagine my life without you. Thank you. Last but not least, I
would like to thank good friends I’ve made at the Goran Lab over the years: Tanya
Alderete, Katherine “Kat” Alexander, Christina Ayala, Dr. Courtney Byrd-Williams,
Sherryl Esplana, Dr. Christianne Lane, Rosa Rangel and Laura Salguero. Thank you all!

iv
TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
ABBREVIATIONS viii
ABSTRACT x
CHAPTER 1: BACKGROUND 1
Chapter 1 Introduction 1
Obesity, diabetes and cardiovascular disease in the Latino population 1
Cardiometabolic risk factors 3
Definition of the metabolic syndrome in children 4
Progression of atherosclerosis 6
Utility and history of CIMT to assess atherosclerosis risk 7
The endothelium: its function and role in atherosclerosis 9
Mechanisms of insulin resistance and hyperglycemia in atherosclerosis
progression 11
Formation of advanced glycation end products 13
Rationale for the study of atherosclerosis risk in children at risk for type 2 diabetes 14
Objectives and specific aims/hypotheses 15
Research Methods 17
Study Sample 17
Power Analysis 19
CHAPTER 2: INTER-RELATIONSHIPS OF CARDIOMETABOLIC RISK FACTORS
AND CAROTID INTIMA MEDIA THICKNESS 23
Chapter 2 Abstract 23
Chapter 2 Introduction 23
Chapter 2 Methods 25
Chapter 2 Results 29
Chapter 2 Discussion 30
CHAPTER 3: PERSISTENCE OF THE METABOLIC SYNDROME AND ITS
INFLUENCE ON CAROTID INTIMA MEDIA THICKNESS 39
Chapter 3 Abstract 39
Chapter 3 Introduction 40
Chapter 3 Methods 41
Chapter 3 Results 43
Chapter 3 Discussion 45
CHAPTER 4: TEMPORAL PATTERNS OF VISCERAL ADIPOSITY AND INSULIN
RESISTANCE AND THEIR EFFECTS ON CAROTID INTIMA MEDIA THICKNESS 54
Chapter 4 Abstract 54
Chapter 4 Introduction 55
Chapter 4 Methods 56
Chapter 4 Results 58
Chapter 4 Discussion 59

v

CHAPTER 5: PROGRESSION OF CAROTID INTIMA MEDIA THICKNESS AND ITS
RELATIONSHIP TO CARDIOMETABOLIC RISK FACTORS 66
Chapter 5 Abstract 66
Chapter 5 Introduction 67
Chapter 5 Methods 68
Chapter 5 Results 73
Chapter 5 Discussion 75
CHAPTER 6: SUMMARY OF FINDINGS, FUTURE DIRECTION, CONCLUSION 84
Summary of chapters 2-5 84
Mechanisms underlying the inter-relationships of hyperglycemia, glucose
effectiveness and CIMT 87
Mechanisms underlying the inter-relationships of high blood pressure,
LDL-cholesterol and CIMT 90
Strengths and associated clinical relevance 91
Limitations of the study design 92
Limitations of the metabolic syndrome definition 95
Future study 98
Conclusion 104

BIBLIOGRAPHY 106

vi
LIST OF TABLES

Table 1-1: Minimum detectable differences in CIMT for each metabolic syndrome
component in preliminary analysis 20

Table 1-2: Minimum detectable differences in CIMT for each metabolic syndrome
component in Paper 1 21

Table 1-3: Minimum detectable differences in various cardiometabolic predictors
for 3 groups CIMT progression in Paper 3 22

Table 2-1: Physical and metabolic characteristics of 120 overweight Latino
Children 34

Table 2-2: Unadjusted correlations between adiposity, measures of glucose
metabolism and insulin action with CIMT 35

Table 2-3: Determinants of CIMT using multivariate regression 38

Table 3-1: Baseline physical and metabolic characteristics by metabolic syndrome
group 49

Table 3-2: Baseline descriptive statistics of the individual metabolic syndrome
components by metabolic syndrome group 50

Table 3-3: Associations between CIMT and the individual components of the
metabolic syndrome at baseline and at time of CIMT measurement 51

Table 4-1: Baseline unadjusted descriptive characteristics by tertiles of CIMT 61

Table 4-2: Baseline unadjusted glucose and insulin action measures by tertiles of
CIMT 62

Table 5-1: Descriptive table of baseline physical and metabolic characteristics by
gender 80

Table 5-2: Determinants of CIMT progression and advanced progression using
multivariate logistic regression 83

Table 6-1: Summary of results from all studies 86

Table 6-2: Criteria for 4 pediatric definitions of the metabolic syndrome 96

Table 6-3: Testing sequence 103

vii
LIST OF FIGURES

Figure 1-1: Causes of death for Latinos in the US 2

Figure 1-2: Cardiometabolic risk factors 3

Figure 1-3: Time line for participant recruitment and data collection 19

Figure 2-1: Systolic blood pressure is significantly correlated with CIMT 36

Figure 2-2: CIMT by metabolic syndrome and impaired fasting glucose status 37

Figure 3-1: CIMT by persistence of metabolic syndrome 52

Figure 3-2: CIMT by persistence of individual metabolic syndrome component 53

Figure 4-1: Distinct temporal patterns for fasting glucose by CIMT tertiles 63

Figure 4-2: Temporal patterns of intra-abdominal adiposity by CIMT tertiles 64

Figure 4-3: Distinct temporal patterns for fasting glucose by CIMT tertiles 65

Figure 5-2: Baseline glucose effectiveness is negatively correlated with CIMT
change 81

Figure 5-3: Participants with advanced CIMT progression had significantly higher
baseline LDL-cholesterol and total cholesterol 82

Figure 6-1: Mean CIMT using 4 different metabolic syndrome definitions,
stratified by number of components 97

viii
ABBREVIATIONS

AGE= Advanced Glycation End Product
ANCOVA= Analysis of Covariance
ANOVA= Analysis of Variance
AUC= Area Under the Curve
BMI= Body Mass Index
CIMT= Carotid Intima Media Thickness
CVD= Cardiovascular Disease
DEXA= Dual Energy X-Ray Absorptiometry
FSIVGTT= Frequently Sampled Intravenous Glucose Tolerance Test
GCRC= General Clinical Research Center
HDL= High Density Lipoprotein
IFG= Impaired Fasting Glucose
IGT= Impaired Glucose Tolerance
IL= Interleukin
LDL= Low Density Lipoprotein
MetS= Metabolic Syndrome
MRI= Magnetic Resonance Imaging
NHANES= National Health and Nutrition Examination Survey
NF-!B= Nuclear Factor Kappa B
NOS= Nitric Oxide Synthase

ix
OGTT= Oral Glucose Tolerance Test
ROS= Reactive Oxygen Species
SD= Standard Deviation
SE= Standard Error
SOLAR= Study of Overweight Latino Adolescents at Risk
VCAM-1= Vascular Adhesion Molecule-1

x
ABSTRACT

Cardiovascular disease is the leading cause of death in the general population.
Arterial inflammation and damage manifests as arterial thickening and is the pathological
basis for atherosclerosis. The first signs of atherosclerosis are thought to begin in
childhood and can be assessed using non-invasive ultrasound measures of the carotid
intima media thicknees (CIMT). Children with any of the traditional cardiovascular risk
factors, such as obesity or the metabolic syndrome may have early signs of
undetectable, or subclinical, atherosclerosis. The purpose of this dissertation was to
investigate subclinical atherosclerosis risk in overweight Latino youth with a family
history of type 2 diabetes. The specific aims of this dissertation were: 1) to evaluate
whether persistence of the metabolic syndrome, a clinically relevant tool, was associated
with CIMT; 2) to examine if temporal changes in visceral adiposity and insulin resistance
contributed to CIMT; and 3) to assess the progression of CIMT over a 2-year period. The
over arching hypothesis was that in a cohort of high-risk children, the metabolic
syndrome and related mechanisms such as abdominal adiposity and insulin resistance
would be associated with increased CIMT.
Participants were recruited from the on-going longitudinal Study of Latino
Adolescents at Risk (SOLAR) Project, which examines type 2 diabetes risk in overweight
Latino adolescents by use of total and regional body composition (via DEXA and MRI),
as well as measures of insulin/glucose metabolism using OGTT and FSIVGTT with
Bergman’s Minimal Modeling.
In paper 1, participants who had persistent metabolic syndrome over a 3-year
period had significantly higher CIMT (0.647±0.018mm) compared to those who never
had the metabolic syndrome, (0.600±0.007mm, p<0.01). In paper 2, participants in the
highest CIMT tertile maintained levels of fasting glucose above 90mg/dL over a 3-year

xi
period prior to CIMT measurement, whereas the low and middle CIMT tertiles had a 3-
4% decrease over time (p<0.05). All participants declined in insulin sensitivity over time
(p<0.05). In paper 3, it was shown that a high variability in the magnitude of CIMT
change exists in growing overweight Latino youth (mean progression: 0.017±0.003mm;
+2.8% and mean regression: -0.019±0.002mm; -3.1%). Baseline LDL-cholesterol was
the sole predictor of CIMT progression and the odds of CIMT progression increased by
3% for each 1 mg/dL higher baseline LDL-cholesterol [95% confidence interval (CI),
1.004-1.006, p=0.03].
In conclusion, measures of insulin action were not associated with CIMT or
progression of CIMT. Elevated baseline CIMT was associated with a 3-year history of
persistent metabolic syndrome, persistent high blood pressure, persistent high waist
circumference and maintenance of fasting glucose levels above 90mg/dL. CIMT
progression that was beyond the physiological norm over a 2-year period (CIMT
>0.100mm) was solely predicted by baseline LDL-cholesterol.

1
CHAPTER 1: BACKGROUND

Introduction
This chapter begins with a brief summary on epidemiological studies of obesity
and cardiovascular disease, which highlights the importance of early detection of
underlying pathology and subclinical stages. Overlapping cardiovascular and metabolic
risk factors that predispose one to cardiovascular disease events and type 2 diabetes is
discussed. Since atherosclerosis is considered the most common pathological process
of cardiovascular disease events, a description of atherosclerosis progression and its
associated endothelial dysfunction is provided. The evidence and rationale for studying
atherosclerosis in a population at risk for type 2 diabetes is also described. This chapter
ends with an outline of the research questions and a summary of the study criteria and a
power analysis used for the studies described in this dissertation.
Obesity, diabetes and cardiovascular disease in the Latino population
According to the 2000 US Census, the Latino population comprised 12.5% of the total
US population.
3
Between 2000 and 2006, Latinos accounted for one-half of the
population growth in the U.S., making them the largest and fastest growing U.S. minority
group. Obesity is a significant problem in Latino adults and recent estimates from the
2007-2008 NHANES that reported 77.9% of Mexican Americans as being classified as
overweight and 37.9% as obese.
58
Figure 1 shows the various causes of death in
Latinos (described as Hispanic in figure) with cardiovascular disease as the number #1
cause of all deaths.

2

Figure 1.2 Cardiovascular diseases are the #1 cause of death in Latinos.
The disproportionate burden of obesity and its related cardiometabolic
consequences has been shown in children as well. Since the year 2000, Latino children
in the United States have higher prevalence of being overweight compared to non-Latino
Caucasian or African-American children.
121-123
In 2008, 41.8% of Latinos ages 12 to19
had a BMI "85
th
percentile for age and height and 23.2% had a BMI "95
th
percentile.
121

More alarmingly, the SEARCH for Diabetes Youth Study showed that Latino youth had
significantly higher odds of having at least two cardiovascular risk factors than any other
ethnic group; older youth (ages 10-19) had significantly higher odds than younger youth
(ages 3-9).
138

Cardiovascular disease risk has also been assessed by prevalence of impaired
fasting glucose ("100mg/dL) using the 1999-2000 NHANES data. These data show that
Latino adolescents had the highest prevalence of impaired fasting glucose compared to
any other ethnic group; subjects with impaired fasting glucose had significantly higher
triglycerides and systolic blood pressure and lower HDL cholesterol when compared to
Latinos with normal fasting glucose subjects.
174
Despite these observations and
associated health risks for cardiometabolic disease, there is a negligible amount of
information on early predictors of atherosclerosis in overweight Latino youth.

3
Cardiometabolic Risk Factors
Type 2 diabetes and cardiovascular disease are concomitant chronic diseases
that share the same risk factors including obesity, hypertension, genetics/family history,
dyslipidemias and the metabolic syndrome (AKA, insulin resistance syndrome as shown
in Figure 1.2). The term cardiometabolic risk represents a clustering of these risk factors
that increase a person’s overall risk for type 2 diabetes and cardiovascular disease.
Assessing cardiometabolic risk requires a comprehensive clinical evaluation that
includes a thorough investigation of family history.

Figure 1.2: Cardiometabolic risk factors
The metabolic syndrome, as coined by Gerald Reaven, defines a constellation of
cardiometabolic risk factors associated to abdominal adiposity and insulin resistance.
134,
135
The five risk factors include: high triglycerides, low HDL-cholesterol, high blood

4
pressure, hyperglycemia and a high waist circumference. In adults, the metabolic
syndrome has been associated with increased cardiovascular disease mortality.
84, 85, 117

In addition, sub-clinical states of glucose intolerance (i.e. pre-diabetes) have also been
associated with 15-54% greater cardiovascular disease mortality rate.
146
Hence, the
sequence of events leading to the onset of cardiovascular disease (including
atherosclerosis) have been linked to hyperglycemia and insulin resistance.
70
The
increased risk for cardiovascular disease has been shown to appear prior to the onset of
type 2 diabetes in adult populations
97
, including Latinos.
71, 104

Definition of the metabolic syndrome in children
In 2001, the National Cholesterol Education Program Adult Treatment Panel
(ATP) III report provided the first standardized definition for the metabolic syndrome in
adults.
7
The syndrome was defined by the ATP III as a combination of 3 or more of the
following conditions: blood pressure "130/85 mm Hg, fasting glucose "110 mg/dl, waist
circumference >102 cm in males and >88 in females, triglycerides "150 mg/dl, or high-
density lipoprotein (HDL) cholesterol #40 mg/dl in men and #50 mg/dl in women.
Recently, an updated cutoff of fasting glucose "100 mg/dl replaced the previous cutoff of
fasting glucose "110 mg/dl to reflect the current American Diabetes Association
definition of impaired fasting glucose.
1

Puberty is associated with hormonal changes that can affect metabolic profiles,
hence the experts in the field have yet to agree on a pediatric definition of the metabolic
syndrome.
152
Our group has proposed a definition for the metabolic syndrome that
applies age-, gender-, and ethnic-specific cut-points to the definition proposed by the
ATP III.
47
According to our definition, the presence of 3 or more of the following
constitutes the metabolic syndrome: waist circumference "90th percentile for age,

5
gender, and ethnicity from NHANES III data,
56
triglycerides "90th percentile of age and
gender,
75
HDL cholesterol # 10th percentile for age and gender,
75
systolic or diastolic
blood pressure >90th percentile adjusted for height, age, and gender,
5
and a 2-hour post
oral challenge plasma glucose value of "140 but < 200mg/dl.
10

Our group conducted an analysis that showed moderate agreement between
three metabolic syndrome definitions.
43
These included the definition proposed by our
group (Cruz et al.
47
), in addition to the one purposed by Cook et al.
43
, and the one
proposed by Weiss et al.
171
The prevalence of the metabolic syndrome ranged from 26-
39% and there was moderate to substantial agreement between definitions (kappa of
0.52-0.70). The variance in estimates may have been due to the less stringent cut-points
for HDL-cholesterol, triglycerides in the Cook et al. definition. The Cook et al. definition
also used impaired fasting glucose as opposed to impaired glucose tolerance. Our group
selected impaired glucose tolerance for the hyperglycemia criteria of the metabolic
syndrome despite the practicality and ease of the fasting glucose measurement. The
reasoning was that in our cohort of overweight Latino youth, those with impaired glucose
tolerance exhibit normal levels of fasting glucose. This suggests that fasting glucose
measures are not as sensitive in detecting pre-diabetes.
64

Since the metabolic syndrome was designed to assess both cardiovascular and
metabolic risk, our research lab has tested the relationship of the metabolic syndrome
definition and cardiometabolic outcomes. First, we have reported that 30% of overweight
Latino children with a family history of type 2 diabetes have the metabolic syndrome.
47

Longitudinal studies of this cohort have shown that children with either persistent
metabolic syndrome or persistent pre-diabetes (having a fasting glucose "100 mg/dL or
a 2-hour glucose "140 mg/dL and <200 mg/dL) had progressively increasing visceral
adiposity, decreasing insulin sensitivity and decreasing beta cell function over time
66, 167
.

6
Studies done outside our laboratory have reported an assocation between the metabolic
syndrome and elevated CIMT.
13
Three other studies of obese children have shown that
insulin resistance, an underlying mechanism of the metabolic syndrome, is an
independent risk factor for increased CIMT.
13, 61, 137
Together, these results suggest that
the metabolic syndrome and insulin resistance have cardiometabolic consequences in
children.
Progression of atherosclerosis
Atherosclerosis is a condition where build-up of cholestrol and fatty materials
accumulate in the intimal and medial layers and cause thickening of medium to large
arteries. The progression of atherosclerosis occurs over many decades and is slowly
progressive and cumulative. Beginning with damage to the arterial wall and the
formation of fatty streaks, the process then leads to the slow formation of atheromas.
29

The next stage of atherosclerosis progression is the formation of atheromas, which are
primarily composed of macrophages and cholesterol crystals. In more advanced stages,
atheromas become complex lesions and calcification may be present. Clinical
atherosclerosis occurs when advanced atheromas or plaques reach a point of
symptomatic manifestation (via plaque rupture resulting in thrombus and/or occlusion of
the artery).
Atherosclerosis has been considered the most common underlying pathological
process of cardiovascular disease and is likely to exist in a subclinical stage for many
decades.
15, 29, 168
Human autopsy studies, such as the Bogalusa Heart study and the
Pathological Determinants of Atherosclerosis in Youth (PDAY) study, have provided
substantial insight on the atherosclerotic process. Fatty streaks were observed in at least
one blood vessel in over 90% of 204 autopised individuals aged 2-39.
22
In addition, the
prevalence of plaques in both aortic and coronoary arteries increased with age. The

7
results from the PDAY study confirmed that atherosclerosis begins in childhood and its
extent and prevalence increases with age.
22
Lesions in the right coronary arteries
progressively increased from 60% in adolescent aged 15-19 years to almost 80% in
adults aged 30-34 years. These studies also showed that cardiovascular risk factors
were related to the early stages of atherosclerosis.
23
Notably, the extent of fatty streaks
increase markedly with multiple risk factors, such as BMI and high blood pressure.
110, 118

The results of these two landmark studies suggest that atherosclerosis appears very
early in life and its severity becomes more apparent with age. Based on these studies,
detection of intima-media thickening at early stages, in conjunction with measurement of
traditional risk factors could yield substantial utility to clinicians in cardiovascular disease
risk assessment.
Utility and history of CIMT to assess atherosclerosis risk
Serial coronary angiography is considered the “gold standard” for measuring
vascular disease progression. Although highly invasive, this method allows visualization
of the vessel lumen and allows for trials of shorter duration compared to clinical trials
that analyze myocardial infarction, stroke, or death as clinical end points.
76
Due to its
expensive and invasive methodology, it is normally reserved for symptomatic patients.
Recently, CIMT has been recognized as a valid surrogate marker for subclinical
atherosclerosis and is amongst various methodologies used in place of serial coronary
angiography. The use of non-invasive measures such as CIMT has become increasingly
important for the early detection of atherosclerosis and possible prevention of coronary
disease events.
Measurement of the aorta arterial wall IMT with high-resolution B-mode
ultrasound imaging was first described in 1986 by Pignoli et al.
129
These investigators

8
performed autopsy studies of excised aortas and were able to demonstrate that the use
of ultrasound image measures had the same thickness measured by microscopy. Since
1986, many improvements on the methods of image acquisition and processing of B-
mode ultrasound images have transpired, namely those by Selzer et al. who developed
a method that utilizes computerized edge tracking-multi-frame image processing.
150
This
technique allows for more accurate measures of CIMT, compared to other
methodologies.
Beginning with Salonen and associates in 1993, many researchers began to
show CIMT as a reliable predictor of coronary disease events. Since then, 13 studies
have shown that elevated CIMT predicts future clinical end points (myocardial infarction,
coronary surgical procedures and/or cardiac death).
20, 37, 38, 93, 120, 145
These populations
were free of any previous cardiac events and 12 of the 13 studies were done in adults
>40 years old. The investigation by Lorenz et al.
103
was the only study to report on a
very large age range (19-90 years of age). Not only did they confirm that CIMT
independently predicted clinical end points, they also observed higher predictive values
(hazard ratios) in the younger patients (<50 years old) than in the older patients (>50
years old). Follow-up time from baseline was relatively short on most studies (<5 years)
and the estimate of association used only had a single measure of CIMT at baseline.
Currently, this is the only study that supports CIMT measurement in a younger
population as a predictor of future cardiovascular disease events.
Progression of CIMT in children and any associated risks have yet to be
determined, therefore data from adults must be extrapolated to younger populations.
The Cholesterol Lowering Atherosclerosis Study (CLAS) assessed CIMT progression
and its relationship to coronary disease events (coronary angioplasty or bypass graft

9
surgery, non-fatal myocardial infraction or coronary death). With a follow-up time of 8-
years, a 0.03 mm increase in CIMT per year translated into to a relative risk of 3.1 for
any coronary event.
78
Although these data cannot be directly applied to children, it is the
only study that provides insight to CIMT progression and associated cardiovascular
disease risk.
The endothelium: its function and role in atherosclerosis
To appreciate the mechanisms of atherosclerosis progression that are
associated with cardiometabolic risk factors, it is important to understand the vital
functions of the endothelium and its resident cells. The endothelium is composed of
three layers: the intima (which is directly exposed to bloodflow), the media (composed of
smooth muscle cells) and finally the adventitia (the outer structural layer composed of
connective tissue). The three main functions of the endothelium are to: 1) to react to
mechanical forces, 2) to serve an anatomical barrier, and 3) to produce signals that
prompt protective actions from toxic substances. Various hypotheses have been
proposed with regard to failure of any of these three endothelial functions and how they
lead to the atherosclerosis process, which will now be discussed.
Mechanical and hemodynamic forces have been proposed and include two main
types: tensile stress and wall shear stress. Tensile stress is a radial and tangential
outward force that is exerted on the vessel wall by the blood pressure. Wall shear stress
is a frictional force that is exerted parallel to the vessel wall directly related to the
viscosity of blood.
48
Acute changes to either tensile or wall shear stress can cause
adjustments in vascular tone while chronic alterations of these forces can lead to a
modification of the arterial walls. For example, tensile stress caused by hypertension is
proposed to have an effect on IMT thickness by increasing the thickness of the medial

10
layer (as opposed to the endothelial layer). Studies have also shown that atherosclerosis
affects the arteries in a site-specific manner, with preference towards the inner wall
curvatures and outer wall bifurcations.
140
It has been proposed that these sites have a
higher vulnerability (i.e., a damaged endothelial layer) due to their exposure to shear
stress and low wall shear rate.
14
Tensile and shear stress are interrelated and have
detrimental effects to the intimal and/or medial layers of the endothelium.
The endothelium acts as an anatomical barrier to shield the inner elastic smooth
muscle layer from any toxic substances circulating in the bloodstream. Besides acting as
a shield, the endothelium synthesizes and regulates various proteins that act as growth
modulators and mediate vascular health.
132
A vital role of these proteins is to maintain an
even surface area by reducing smooth muscle cell proliferation into the intima. Landmark
studies by Russell Ross and John Glomset were the first to show that certain blot clots
contained a growth factor responsible for smooth muscle proliferation when compared to
plasma serum, in vitro.
141
The growth factor is now known as “platelet-derived growth
factor” (PDGF). In 1976, Ross and Glomset published their “response-to-injury
hypothesis”, proposing that injury (mechanical, chemical or immunologic) to the
endothelium would lead to platelet aggregation via PDGF.
142, 143
Further studies by Ross
described various factors that may contribute to injury of the endothelium, including
hyperlipidemias, homocysteinemia, hypertension, infection, or other pro-inflammatory
cytokines.
140

Nitric oxide is another essential molecule synthesized in the endothelial cells. It is
derived from arginine and oxygen by nitric oxide synthase (NOS).
111
Nitric oxide has
three important functions: 1) it causes vasodilation of vascular smooth muscle, and 2)
mediates molecular signaling pathways that prevent platelet and leukocyte interaction

11
and 3) inhibits vascular smooth muscle cell proliferation. Hence, the bioavailability of
nitric oxide is important in vascular health, as the lack of nitric oxide has been shown to
increase proinflammatory factors, such as IL-6, IL-1, TNF-$, and NF-!B, in cultured
human cells.
177
This leads to the expression of leukocyte adhesion molecules (such as
monocyte adhesion molecules) via the up-regulation of vascular adhesion molecule-1
(VCAM-1), ultimately causing the accumulation of foam cells and setting the foundation
of atheromas.
The accumulation of macrophages/monocytes is detrimental due to their
conversion by oxidized LDL-cholesterol to the characteristic “foam cell”. Nikolai
Anitschokow first discovered foam cells, during his experiments with cholesterol-fed
rabbits and he correctly speculated that “cholesterol-loaded cells” were actually white
blood cells entering the artery wall.
158
His ideas were not initially accepted, but his work
was the basis for the lipid hypothesis of atherogenesis. The lipid hypothesis has
gradually developed over many decades, beginning with the characterization of
lipoproteins, to the discovery of the LDL-cholesterol receptor and scavenger receptors
on macrophages by Nobel Prize winners Michael Brown and Joseph Goldstein.
157
In the
early 1980s, several researchers noted that LDL-cholesterol existed in modified forms
that were denser and more electronegative. These characteristics made this type of
LDL-cholesterol (oxidative LDL) into a more atherogenic form. Here arose the now
generally accepted “oxidative modification hypothesis”, where oxidized LDL-cholesterol
crosses the endothelial layer (through the normal phagocytotic nature of macrophages)
to contribute to the formation of foam cells and eventually atheromas.
157

Mechanisms of insulin resistance and hyperglycemia in atherosclerosis progression
The link between type 2 diabetes and cardiovascular disease events has been

12
clearly shown in epidemiological studies.
19, 113, 166
Insulin resistance and hyperglycemia
have both been associated with endothelial dysfunction, which is a precursor to the
vascular changes leading to cardiovascular disease events, such as myocardial
infarction and stroke. The major physiological mechanisms underlying these
relationships include the roles of elevated free fatty acids, alterations in insulin signaling
transduction pathways, increased oxidative stress, advanced glycation end products
(AGEs) and increased nuclear factor kappa B (NF-kB).
45, 105, 107
These mechanisms are
thought to have further molecular consequences on the availability of various endothelial
factors, primarily the bioavailability of nitric oxide, which affects the vasodilation and
inflammatory processes that ultimately lead to atherosclerosis.
Insulin resistance normally precedes and then coincides with hyperglycemia and
type 2 diabetes. Studies in humans have demonstrated that insulin resistance is often
associated with endothelial dysfunction.
11, 159, 165
Insulin-activated nitric oxide synthase
(NOS) production (as part of the phospatidylinositol-3 kinase pathway (PI3K)) triggers
production of nitric oxide by the endothelial cells. Hence, in healthy subjects, insulin
would increase endothelial vasodilation but in insulin-resistant subjects this vasodilation
has been shown to be reduced.
96
The decrease of nitric oxide as a consequence of
alterations of the insulin signaling transduction (or the PI3K pathway) prevents the
activation of NOS and therefore, decreases proper vasodilation and contributes to
arterial thickening.
Insulin resistance of adipose cells has also been associated with increased
lipolysis and subsequent elevated circulating free fatty acids, causing hepatic insulin
resistance (The Portal Theory).
25, 28
Visceral adipose tissue, which is highly insulin
resistant, releases more free fatty acids compared to other types of adipose tissue.
173

13
High levels of circulating plasma free fatty acids can impair endothelial function
by increasing production of free radicals and by activating protein kinase C (PKC). PKC
has been shown to decrease insulin receptor substrate-1 in the PI3K pathway.
53, 69
The
result is decreased NOS activity, reduced production of nitric oxide, and decreases in
endothelial function. In addition, the resulting hepatic insulin resistance, caused by
elevated free fatty acids can stimulate hepatic gluconeogenesis and could lead to
hyperglycemia and glucose intolerance.
57

Hyperglycemia causes oxidative stress, which is a state of imbalance caused by
the production of reactive oxygen and the body’s ability to repair any resulting damage.
Reactive oxygen species (ROS), produced from peroxides, are the most destructive free
radicals. In diabetic models, the prominent ROS is superoxide anion, which is known to
inactivate nitric oxide to form peroxyntirite and then initiate a cascade of events resulting
in a continuous production of superoxide anion and inactivation of nitric oxide.
45

Hyperglycemia is thought to initiate this chain by increasing superoxide anion production
via the electron transport chain in the mitochondria.
119
This overproduction of superoxide
anion is also implicated in increased intracellular production of AGEs, which are
modifications of lipids (or proteins) caused by non-enzymatic oxidation or glycation after
contact with aldose sugars.
63

Formation of advanced glycated end-products
The formation of AGEs contributes to vascular disease pathophysiology.
34, 139

Various factors are involved in the formation of AGEs including the degree of
hyperglycemia and the extent of environmental oxidative stress.
148
When these
conditions are present, proteins may become either oxidized or glycated. The
biochemical process responsible for AGE formation is the Maillard reaction, which

14
requires Schiff bases and the Amadori product, which is a highly reactive carbonyl
group. The Amadori product is derived from the reaction of a carbonyl group of a
reducing sugar with proteins or lipids.
33
During Amadori reorganization, a build-up of
these products occurs, known as “carbonyl stress”, during which $-dicarbonyls can react
with functional groups (amino, sulfhydryl, and guanidine) in proteins and result in
denaturation or cross-linking. In addition, $-dicarbonyls also react with the lysine and
arginine functional groups and leads to a stable AGE formation.
33, 63
AGEs are virtually
irreversible and can bind to several types of receptors. The most common, RAGE, will
recognize and bind AGEs, initiating intracellular signaling and disrupting normal cellular
function.
The accumulation of AGEs can affect vascular cell function by modifying the
extracellular matrix and by interfering with hormonal and free radical function by
engaging cell surface receptors.
34
A detrimental example of AGEs is when they become
cross-linked to lipids in LDL-cholesterol. This has been shown in cholesterol samples of
patients with and without diabetes.
35
Glycated and/or oxidative LDL-cholesterol have
also been shown to reduce production of nitric oxide; when bound to receptors on
endothelial cells, it suppresses the clearance of LDL-cholesterol.
60, 131

Rationale for study of subclinical atherosclerosis risk in children at risk for type 2
diabetes
The pathophysiology of diabetes and atherosclerosis has been related to
traditional cardiometabolic risk factors, as well as insulin and glucose mechanisms. In
this dissertation, we want to ascertain if insulin resistance and associated hyperglycemia
and increased visceral adiposity, play a role in the thickening of the carotid artery of
overweight Latino youth at with a family history of type 2 diabetes.

15
There is some debate as to whether atherosclerosis begins at glucose levels
below the threshold of type 2 diabetes onset. A meta-analysis of 20 studies and over
90,000 participants showed a positive relationship between fasting glucose and 2-hour
glucose with the relative risk of CVD events.
44
Furthermore, the elevated risk for a CVD
event extended below the current thresholds of impaired fasting glucose (100mg/dL) and
impaired glucose tolerance (140mg/dL). Due to lack of data on traditional risk factors
(such as lipids and blood pressure) in all 20 studies, this analysis could not be adjusted
for these factors. Therefore, some doubt remains with regard to glucose as an
independent predictor of CVD events. Nevertheless, this study does support the notion
that macrovascular disorders may begin well below the threshold of overt diabetes. This
dissertation is a unique opportunity to test the early onset of atherosclerosis in
overweight children who are highly insulin resistant and therefore more likely to exhibit
elevated glucose levels.
Objectives and specific aims/hypotheses:

The purpose of this dissertation was to assess sub-clinical atherosclerosis risk in
overweight Latino adolescents with a family history of type 2 diabetes. The overarching
hypothesis is that in this well-defined cohort of high-risk children, the metabolic
syndrome and related mechanisms such as abdominal adiposity and insulin resistance
are associated with increased CIMT. A preliminary analysis (results shown in Chapter 2)
showed that the metabolic syndrome, visceral adiposity and insulin sensitivity were not
associated with CIMT in overweight Latino children. However, children with impaired
fasting glucose did have higher CIMT than those with normal fasting glucose and
systolic blood pressure was the independent predictor of CIMT. Using these results in
conjunction with the literature, the proposed dissertation papers with specific aims and
hypotheses were defined as follows:

16
Paper 1: Persistence of the metabolic syndrome and its influence on carotid artery
intima media thickness
! Specific Aim 1: To determine the effects of persistent metabolic syndrome and
the individual metabolic syndrome components over a 3-year period on CIMT.
! Hypothesis 1: Participants with persistent metabolic syndrome will have
increased CIMT compared to those who never had the metabolic syndrome.
Paper 2: Temporal patterns of visceral adiposity and insulin resistance and its
influence on carotid intima media thickness
Specific Aim 2: To examine the history of temporal patterns of abdominal adiposity and
insulin resistance that influence CIMT.
! Hypothesis 2: Children with increasing visceral adiposity and declining
insulin sensitivity will have higher CIMT.
Paper 3: Progression of subclinical atherosclerosis

! Specific Aim 3: To examine the progression of CIMT and its cardiometabolic
predictors that contribute to CIMT change over a 2-year period.
! Hypothesis 3: The development of sub-clinical atherosclerosis will be
characterized by a decrease in insulin sensitivity and an increase in visceral
adiposity, systolic blood pressure and fasting glucose.

17
Research Methods

Study Sample
The data used in the series of studies contained in this dissertation were
collected from the longitudinal study entitled, Study of Overweight Latino Adolescents At
Risk (SOLAR). This section describes the specific inclusion and exclusion criteria for all
participants included in these analyses. Specific procedures and methodology are
described in the methods section of each chapter.
All participants who met the study enrollment requirements signed informed
consent and child assents. Once enrolled, participants were called annually and invited
to come for outpatient and inpatient visits at the University of Southern California (USC)
General Clinical Research Center (GCRC). Due to budget cuts, in the 2004 calendar
year, we only invited families to attend one visit for an abbreviated version of the full
study protocol. The original enrollment protocol is described below.
The children were recruited from the community surrounding Los Angeles
County/USC Hospital in East Los Angeles from the years 2001 to 2003. The majority of
children (80%) were recruited from various pediatric clinics, and the remainder by word-
of-mouth (i.e., contacts obtained from families already in the study) and local advertising.
Inclusion criteria: Only children with a body mass index greater than the 85
th

percentile for age and sex at the first visit to the study were included in the cohort. The
next inclusion criteria was the presence of a positive family history of type 2 diabetes,
which was defined as the presence of diagnosed type 2 diabetes of at least one parent,
grandparent, or sibling at inception of the study. Children were defined as Latino if they
and both parents and both sets of grandparents identified with this cultural group.
Approximately 90% of the children were from Mexico; the remainder were from Central
America.

18
Exclusion criteria: All children were screened with an OGTT to ensure they were
free of type 1 and type 2 diabetes. Ineligible children included those with symptoms of
polyuria, polydipsia with or without unexplained weight loss, fasting plasma glucose >
126 mg/dl, or a 2-hour plasma glucose >200 mg/dL during an oral glucose tolerance
test. Children with impaired glucose tolerance (IGT, 2-hour plasma glucose >140 mg/dl
during an OGTT) and/or conditions associated with insulin resistance (e.g. acanthosis
nigricans, hypertension, dyslipidemia, polycystic ovarian syndrome) were not excluded.
If at any time a child was diagnosed with type 2 diabetes, a repeat metabolic
characterization was performed and the family was referred for treatment. All subjects
were screened by a medical history evaluation and physical examination. Subjects were
not eligible for the study if they were: a) taking any medications known to influence body
composition or insulin action or insulin secretion (e.g. prednisone, ritalin, growth
hormone); b) diagnosed with syndromes or diseases that may influence insulin action
and secretion (e.g. maturity-onset diabetes of the young, lipoatrophic diabetes, cystic
fibrosis), or body composition and fat distribution (e.g. Cushing syndrome, Down
syndrome); or, c) previously diagnosed with any major illness (e.g. severe intrauterine
growth retardation, chronic birth asphyxia, cancer).
Once participants were recruited and qualified for the SOLAR study, they
attended annual visits for full cardiometabolic profiling including glucose and insulin
measures, blood pressure, a full physical examination with maturation staging, and full
body and abdominal adiposity measures. The timeline (Figure 1-3) shows that the first
CIMT was collected in 2006 (the baseline measure) and for those participants who
attended in 2008 a follow-up CIMT was performed.

19
Figure 1-3: Timeline of recruitment and data collection

Power and sample size analysis
Statistical power analysis is essential to evaluate the number of subjects needed
to detect an effect in the relationship between any of the cardiometabolic risk factors and
CIMT. The minimum detectable mean differences or associations were calculated to
determine how the sample sizes of the data collected on CIMT might have affected the
ability to detect the hypothesized effects. For the purposes of this dissertation, the
minimal detectable effects were calculated for correlations, linear regression and
independent t-tests.
For correlation and linear regression analyses, the minimal detectable difference
represents the smallest coefficient estimate that would be statistically significant given
the specified alpha and power. Alpha, or the type I error probability, represents the
probability that the null hypothesis will be incorrectly rejected. Power is the probability of
correctly rejecting the null hypothesis. Using the sample sizes available for study from
SOLAR, minimum detectable effects were calculated using a two-sided alpha of 0.05
and a power of 0.80. The standard deviation used to calculate the effect sizes was

20
0.50mm. For the cross-sectional analysis where the sample size was 120 participants,
the minimum detectable r-value was 0.25 for unadjusted analysis and for our follow-up
analysis of 72 participants, the minimum r-value was 0.26.
The calculated absolute values of the minimum detectable differences were
calculated for analyses (involving t-tests and ANOVA) and are shown in Tables 1.1 –
1.4. In chapter 6 of this dissertation, a post-doc analysis of achieved power will also be
discussed. Power analyses were conducted with G*Power 3.1 (for Mac).

Table 1.1 Minimum detectable difference in group means for each metabolic
syndrome component with CIMT in preliminary analysis (Chapter 2)
Dichotomized groups for independent t-
tests
Minimum detectable difference in
CIMT (mm)
Presence of the metabolic syndrome
(yes/no; 28/92)
0.037
Presence of high blood pressure
(yes/no; 23/97)
0.039
Presence of high waist circumference
(yes/no; 60/60)
0.031
Presence of high triglycerides
(yes/no; 22/98)
0.040
Presence of low HDL-cholesterol
(yes/no; 70/50)
0.031
Presence of impaired glucose tolerance
(yes/no; 19/101)
0.043
Presence of impaired fasting glucose (yes/no;
14/109)
0.048

21

Table 1.2 Minimum detectable CIMT difference in group means for each
metabolic syndrome component in Paper 1 (Chapter 3)
Minimum detectable CIMT difference (in mm)

Between
NEVER and
INTERMITTENT
groups
Between
INTERMITTENT
and
PERSISTENT
groups
Between
NEVER and
PERSISTENT
groups
Persistence of
metabolic syndrome
groups
0.037
(n= 53, 28)
0.050
(n= 28, 16)
0.045
(n= 53, 16)
Persistence of high
blood pressure groups
0.035
(n= 50, 35)
0.054
(n= 35,12)
0.051
(n= 50, 12)
Persistence of high
waist circumference
groups
0.045
(n= 25, 25)
0.039
(n= 25, 47)
0.039
(n= 25, 47)
Persistence of high
triglyceride groups
0.038
(n= 57, 26)
0.053
(n= 26,14)
0.047
(n= 57, 14)
Persistence of low HDL-
cholesterol groups
0.043
(n= 25, 30)
0.038
(n= 30,42)
0.040
(n= 25, 42)
Persistence of impaired
glucose tolerance
groups
0.023
(n= 52, 37)
0.062
(n= 37, 8)
0.060
(n= 52,8)
Persistence of impaired
fasting glucose groups
0.068
(n= 67, 25)
0.080
(n= 25, 5)
0.073
(n= 67, 5)

22

Table 1.3 Minimum detectable difference in group means for 3 groups of CIMT
progression in Paper 3 (Chapter 5)
Minimum detectable difference
Baseline independent
variable
Between Non-
progressor and
normal
progressor
groups
(n=34,15)
Between Normal
progressors and
advanced
progressor
groups
(n=15,23)
Between Non-
progressor and
advanced
progressor
groups
(n=34,23)
Baseline Insulin
sensitivity
((x10
-4
/min
-1
)/µU/mL))
0.78 0.96 0.77
Baseline systolic blood
pressure (mmHg)
7.04 8.60 6.93
Baseline Visceral
adiposity (cm
2
)
14.86 18.16 14.63

23
CHAPTER 2: INTER-RELATIONSHIPS OF CARDIOMETABOLIC RISK
FACTORS AND CIMT (PRELIMINARY ANALYSES)

Chapter 2 Abstract
The relationships between cardiovascular risk factors and measures of
subclinical atherosclerosis have been extensively studied in adults but there is sparse
data in children, especially minority populations. This cross-sectional analysis included
120 healthy male and female overweight Latino adolescents (mean age: 14.6 yrs; mean
BMI percentile: 96.7). High resolution B-mode ultrasound was used to measure CIMT.
Body fat and lean tissue mass were measured by DEXA and visceral and subcutaneous
adipose tissue by MRI. Insulin sensitivity was measured by an intravenous glucose
tolerance test. There were no significant associations between adiposity, insulin
sensitivity, metabolic syndrome or traditional cardiovascular risk factors and CIMT.
Children with impaired fasting glucose had higher CIMT before and after controlling for
covariates (means ± SE; 0.638 ± 0.015 vs. 0.609 ± 0.001 mm, p=0.03). Multivariate
linear regression revealed that systolic blood pressure was an independent predictor of
CIMT. These findings suggest that young overweight Latinos may exhibit subclinical
atherosclerosis through these adiposity-related comorbid risk factors for vascular
disease.
Chapter 2 Introduction
The increasing prevalence of pediatric childhood obesity
59
warrants investigation
of the link between obesity and atherosclerosis risk in youth. The Latino population in the
US is rapidly growing and has shown a variety of obesity-related comorbidities, such as
hypertension and diabetes, placing them at high-risk for cardiovascular disorders.
Overweight Latino children have a high prevalence of obesity
59
, insulin resistance
65
,

24
metabolic syndrome
47
and impaired fasting glucose/pre-diabetes
138
, all of which may
contribute to the early development of atherosclerosis.
Carotid artery intima media thickness (CIMT) is a noninvasive measure of
subclinical atherosclerosis. Studies in adults have shown these measures to be
significantly correlated with adiposity, diabetes, and traditional vascular risk factors such
as lipids and blood pressure. Evidence suggests that the metabolic syndrome is
associated with cardiovascular disease and CIMT.
8
Comparable research studies in
youth are sparse but those that have been performed show similar relationships to those
in adults. Several case-control studies have reported increased CIMT in overweight
children compared to their normal weight counterparts.
136, 175, 178
Investigators have also
shown an association of CIMT with dysfunction in glucose metabolism
13, 137
,
hypercholesterolemia
27
and hypertension
156
. In overweight youth, the metabolic
syndrome has been correlated to CIMT.
137

Although the relationship of CIMT with vascular disease risk factors has been
demonstrated in adults and some pediatric studies, the literature is deficient in studies
involving overweight Latino youth. Therefore, the first aim of this study was to examine
the relationships of metabolic correlates, including adiposity and measures of glucose
metabolism dysfunction to CIMT and arterial stiffness in a cohort of overweight Latino
youth. The second aim was to investigate the associations of the metabolic syndrome
and its individual components with CIMT. It was hypothesized that visceral adiposity,
insulin resistance, the metabolic syndrome and its various components would be
associated with CIMT.

25
Chapter 2 Methods

Participants: This study included data from an ongoing cohort study (SOLAR:
Study of Latino Adolescents At Risk) exploring metabolic risk factors for type 2 diabetes
in Latino children. Study participants satisfied the following criteria for inclusion at first
visit: age- and gender-specific BMI " 85
th
percentile, 8-13 years of age, positive Latino
ethnicity (i.e., parents and grandparents of Latino descent), positive family history for
type 2 diabetes, and absence of diabetes as assessed by oral glucose tolerance test
(OGTT). Participants were excluded if they were using a medication or diagnosed with a
condition known to influence body composition or insulin / glucose metabolism. Although
subclinical atherosclerosis measures were collected on 122 subjects only those with
complete measures of adiposity, insulin resistance and the metabolic syndrome were
included in these analyses (n=120). Prior to any testing procedure, informed written
consent from parents and assent from the children were obtained. This investigation was
approved by the Institutional Review Board of the University of Southern California.
Study Protocol: Methods from the SOLAR protocol have been previously
reported in detail.
47, 65
A comprehensive medical history and physical examination was
performed at the annual screening exam for diabetes. This visit included height (by
stadiometer), weight, anthropometry (waist and hip measurements), seated blood
pressure (three measures), and Tanner staging completed by a licensed health care
provider. Following the exam, an OGTT was performed to determine eligibility for the
study. Subjects ingested 1.75 g oral glucose solution / kg body weight (to a maximum 75
g). Blood was sampled and assayed for glucose and insulin at –5 min (“fasting”) and 120
min (“2-hour”). Two-hour insulin and glucose area under the curve (AUC) and
incremental area under the curve (IAUC) were calculated from the OGTT data, in

26
mg/min/dl for glucose and %U/min/ml for insulin. Glucose and insulin AUC are calculated
as the sum of the area of each time segment by insulin or glucose concentration, and
IAUC as the sum of the same area adjusted for the starting point.
Approximately 7-14 days following the screening visit, participants were admitted
for an overnight inpatient hospital visit. Body composition (total fat mass and total lean
tissue mass) was determined by a whole-body dual-energy x-ray absorptiometry (DEXA)
scan using a Hologic QDR 4500W (Bedford, MA). Central fat distribution was measured
directly by magnetic

resonance imaging (MRI) using a General Electric 1.5

Signa LX-
Echospeed device with a 1.5-Tesla

magnet (Waukesha, WI).

A single-slice axial TR
400/16 view of the abdomen at the level

of the umbilicus was analyzed for cross-
sectional area of visceral adipose

tissue and subcutaneous adipose tissue.
144
The
frequently sampled intravenous glucose tolerance test (FSIVGTT) was performed after
an overnight fast as previously described.
47, 65
Glucose and insulin concentrations were
then entered into MINMOD MILLENNIUM 2003 software (version 5.16) for calculation of
insulin sensitivity, acute insulin response (a measure of insulin area under the curve
above basal for the first 10 min of the FSIVGTT), and disposition index (the product of
insulin sensitivity and acute insulin response, an index of beta cell function). Fasting
lipids were assessed using Vitros Chemistry DT Slides (Johnson and Johnson Clinical
Diagnostics, Inc, Rochester, New York).
Subclinical atherosclerosis was measured at the USC Atherosclerosis Research
Unit Core Imaging and Reading Center. High resolution B-mode ultrasound images were
obtained using a Seimens Acuson CV70 (7-14 MHz linear array) imager for CIMT
measures according to the methods of Hodis, et al.
78, 79, 94, 149, 150, 176

27
Metabolic syndrome and individual components: Using a definition similar to the
Adult Treatment Panel (ATP)
2
that we adapted for pediatric populations, the metabolic
syndrome was defined by the presence of at

least three of the following components:
• abdominal obesity (waist circumference " 90th percentile for age, gender and
Hispanic ethnicity)
56
,
• hypertriglyceridemia (triglycerides "90th percentile for age and gender)
75
,
• low HDL cholesterol (HDL cholesterol "10th percentile for age and gender)
75
,
• high blood pressure (either systolic or diastolic blood pressure >90th percentile
adjusted for height, age, and gender)
6
,
• impaired glucose tolerance (OGTT 2-hour glucose: 140 -199 mg/dl)
4
.
An additional hyperglycemia category considered in other pediatric definitions of the
metabolic syndrome is impaired fasting glucose (IFG) which is defined as a fasting
glucose value of 100 -125 mg/dL
4
. This category was not used for the pediatric definition
of the metabolic syndrome, but instead used for the analyses comparing individual
metabolic features with CIMT.
Statistical Analyses: Prevalence of individual components of the metabolic
syndrome was calculated as the total number of participants with each component
expressed as a percentage of the total sample. For the preliminary analyses,
participants were dichotomized into two groups: the metabolic syndrome group included
children with three, four or five features of the metabolic syndrome and the non-
metabolic syndrome group included children with less than three features.
Independent t-test analyses with Levene’s test for equality of variances, were
performed to test mean descriptive and metabolic differences between the gender

28
groups. A chi-square test was performed to compare gender and metabolic syndrome
distributions.
Simple and partial correlations were used to assess the relationship of adiposity
and insulin parameters with CIMT. Linear regression models were then used to explore
whether gender significantly modified the relationship between each of the
cardiovascular risk factors and CIMT, while adjusting for age and height. The normality
and homoscedasicity of residuals in each regression model were evaluated with the
Shapiro-Wilk test and by examining scatter plots. Non-normally distributed variables
were then log transformed and the distribution of the residuals was reassessed to assure
normality.
To compare mean CIMT differences between groups with or without the
metabolic syndrome, independent t-tests and analysis of covariance (ANCOVA)
procedures were used with a Tukey adjustment for multiple comparisons. The
distributions of the residuals of the ANCOVA models were also checked for normality.
Multivariate regression was used to assess independent predictors of CIMT. The
model included age, male gender, height, HDL-cholesterol, triglycerides, waist
circumference, fasting glucose, 2-hour glucose, and systolic blood pressure. Data were
analyzed using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL), with an a
priori 2-sided significance level of p<0.05. Data reported are mean ± standard deviation
(SD).

29
Chapter 2 Results
Table 2-1 lists the descriptive statistics of the 120 participants, which was
composed of 71 males and 49 females. Tanner stage for males ranged from 1-5,
whereas in females the range was limited to the later pubertal stages 3-5 (p<0.05).
Males were significantly taller, heavier and had higher total lean tissue mass compared
to females (p<0.05). The metabolic syndrome was more prevalent in males than in
females (31% vs. 12%, p<0.05). Systolic and diastolic blood pressures were higher in
males compared to females (p<0.05). HDL-cholesterol was significantly lower in males
than in females (p<0.05). Males had higher fasting glucose and hemoglobin A1c
compared to females but females had a lower disposition index (p<0.05). CIMT did not
differ by gender but females had a lower maximum carotid diameter than males
(p<0.05).
There were no significant correlations between CIMT and the measures for
adiposity or glucose/insulin metabolism (insulin AUC, insulin sensitivity, acute insulin
response, and disposition index) (Table 2-2). Other cardiometabolic risk factors (fasting
and 2-hour glucose, waist, triglycerides and HDL- and LDL- cholesterol) were not
correlated with CIMT (data not shown). Systolic blood pressure was significantly
correlated to CIMT after controlling for gender, age, Tanner and height (r=0.21, p<0.05,
Figure 2-1). There were no gender interactions with systolic blood pressure or any
other cardiometabolic risk factor and CIMT (data not shown).
Figure 2-2 shows that the metabolic syndrome had no significant association
with CIMT but participants with impaired fasting glucose had significantly higher CIMT
than those with normal fasting glucose (estimated marginal means ± SE: 0.637 ± 0.014
vs. 0.609 ± 0.006 mm, p=0.03). The multivariate regression model with the metabolic

30
syndrome features showed that gender and systolic blood pressure were independent
predictors of CIMT (Table 2-3, p<0.05).
Chapter 2 Discussion
This study examined cardiometabolic correlates of CIMT indices in an overweight
Latino adolescent cohort. Impaired fasting glucose status was an important correlate of
CIMT, and systolic blood pressure was the sole independent predictor of CIMT. These
findings suggest that impaired fasting glucose and systolic blood pressure may be
important components in atherosclerosis risk assessment of overweight Latino
adolescent youth.
To our knowledge, there have been no prior reports of cardiometabolic correlates
with CIMT in a pediatric Latino population. However, the prevalence of traditional
cardiovascular disease risk factors has been studied and reported by other investigators.
NHANES 1999-2000 data showed that Latino adolescents had the highest prevalence of
impaired fasting glucose of any ethnicity included in that survey and that this same sub-
group had significantly higher systolic blood pressure and lower plasma HDL-cholesterol
levels than normal fasting glucose subjects.
174
The SEARCH for Diabetes Youth Study
showed that Latino youth had significantly higher odds of having at least two
cardiovascular risk factors when compared to other ethnic groups
138
In addition, the
older youth group (ages 10-19) had significantly higher odds of cardiovascular risk than
children (ages 3-9). These data indicate that Latino youth may have an accelerated risk
of developing vascular disease.
A growing body of evidence in children has shown that obesity is a risk factor for
the early development of vascular disorders. Several studies of overweight children have
reported higher mean CIMT compared with their lean matched counterparts.
18, 81, 82, 89, 95,

31
109, 138, 178
This indicates obesity as a strong correlate of CIMT in children. However, none
of these investigations used precise body composition measures for adiposity. Only one
study by Schiel et al.
147
utilized measures beyond BMI in a group of overweight children.
They found that both BMI and percent body fat were significantly higher in those children
with the highest CIMT, compared to those with the lowest CIMT. Our current findings did
not concur although a plausible explanation could be our sample only included
overweight/obese children (mean 96
th
percentile) thereby limiting our study. The
aforementioned articles on pediatric subjects of non-European descent report CIMT
measures ranging between 0.62mm and 0.68mm
95, 178
and those of European descent
ranging from 0.37mm to 0.49mm. Ethnic differences have been previously implicated in
the role of atherosclerosis risk in adults.
101
Although the current results are consistent
with these previous studies in populations of non-European descent, CIMT values
cannot be directly compared due to differing methodologies. Therefore, further research
of multi-ethnic groups in children is needed to conclude that ethnic differences exist.
Dysfunction of glucose and insulin metabolism along with the metabolic
syndrome is associated with atherosclerosis and vascular disease in adults. In the
pediatric literature, CIMT has been related to insulin resistance
13, 61
and impaired
glucose tolerance.
137
Given this, a relationship between CIMT and the metabolic
syndrome was expected, yet this was not the case. Iannuzzi et al.
81
reported no
difference in CIMT by metabolic syndrome status, which agrees with our current
findings. Reinehr et al.
137
analyzed several pediatric definitions of the metabolic
syndrome and CIMT. Among four proposed pediatric definitions of the metabolic
syndrome, only two of these (Viner et al
169
, and Weiss et al.
171
) were related with CIMT.
The investigators determined that the top predictor of increased CIMT in children was

32
impaired glucose tolerance and that the top predictive clustering of risk factors included
high waist circumference, hypertension and impaired fasting glucose. Similarly, the
present study found blood pressure and impaired fasting glucose to be correlates to
CIMT. Further analysis revealed that of the 18 participants with high blood pressure,
61.1% (n=11) also had a high waist circumference. Of the 11 participants with both high
BP and high waist circumference, three also had impaired fasting glucose. Notably,
these three subjects were in the highest quintile of CIMT (>0.660mm). The present study
is consistent with that of Reinehr et al.’s report
137
of pertinent metabolic correlates of
CIMT in overweight youth. Clustering of hypertension, abdominal adiposity, and
impaired fasting glucose may be relevant in the further study of elevated CIMT in
children, especially as targets for the reduction of atherosclerosis in youth.
In adults, CIMT progression has been documented to progress at an average
rate of 0.005 mm/year. The approximate 0.03 mm greater CIMT in the high blood
pressure and impaired fasting glucose group represents a vascular age six years
advanced compared with those with normal blood pressure and fasting glucose. In
adults, the clinical relevance of a 0.03mm increase in CIMT per year translates into to a
relative risk of 3.1 for any coronary event.
78
Although this risk ratio cannot be directly
applied to the pediatric population, it does suggest a carotid intima thickening that may
have clinical relevance.
The strengths of this study include the use of subclinical atherosclerosis
measures with a single sonographer and reader for the ultrasound images. In addition,
accurate measures of total and regional body composition (DEXA and MRI scans) and
direct measures of insulin sensitivity (FSIVGTT with minimal modeling) were utilized
instead of less sophisticated methods (such as BMI, HOMA and QUICKI). The large

33
homogeneous sample of understudied minority youth also contributes to the strengths of
this report. Several limitations are also noteworthy. This design was cross-sectional and
the sample had a very narrow range of BMI. There were no lean participants to use as
matched controls and as a consequence, this limited the findings. Finally, the data
results can only be generalized to the very distinct group of overweight Latino
adolescents with a family history of type 2 diabetes.
In conclusion, systolic blood pressure and impaired fasting glucose were
correlates of CIMT. However, other traditional risk factors such as obesity, insulin
resistance and the metabolic syndrome were not associated with CIMT.

34

Table 2-1: Physical and metabolic characteristics of 120 overweight Latino
children

Males Females p-value
(n= 71) (n= 49)
Age (years) 14.7 ± 1.6 14.7 ± 1.9 NS
Maturation stage (by Tanner)
1/2 12 0 0.003
3 14 11
4/5 45 38
Body composition
Height (cm) 168.3 ± 9.0 159.0 ± 6.0 <0.001
Weight (kg) 88.5 ± 20.8 81.0 ± 20.3 0.049
BMI (kg/m
2
) 31.1 ± 6.0 31.8 ± 6.5 NS
BMI z-score 1.99 ± 0.59 1.89 ± 0.59 NS
Total Lean Tissue Mass (kg) 55.3 ± 11.1 45.0 ± 8.9 <0.001
Total Fat Mass (kg) 28.1 ± 10.8 31.0 ± 11.3 NS

Cardiometabolic risk factors
Presence of metabolic syndrome 22 (31%) 6 (12%) 0.028
Waist Circumference (cm) 94.4 ± 12.7 91.2 ± 13.3 NS
Systolic blood pressure (mmHg) 118.1 ± 9.9 109.8 ± 8.8 <0.001
Diastolic blood pressure (mmHg) 66.7 ± 6.3 62.7 ± 5.8 <0.001
HDL Cholesterol (mg/dL) 35.4 ± 6.9 39.5 ± 8.9 0.004
Triglycerides (mg/dL)
106.9 ± 55.5 95.9 ± 44.7 NS
LDL-cholesterol (mg/dL) 86.0 ± 21.0 81.4 ± 21.6 NS
Total cholesterol (mg/dL) 142.8 ± 25.7 140.0 ± 24.8 NS

Fasting Glucose (mg/dL) 90.4 ± 7.8 85.9 ± 9.8 0.006
2-hr Glucose (mg/dL) 116.7 ± 23.1 120.3 ± 21.1 NS
HbA1c (%) 5.4 ± 0.3 5.2 ± 0.3 0.02
Fasting Insulin (µU/mL) 14.3 ± 9.4 14.1 ± 7.3 NS
Insulin AUC (nmol/min/L) 288.9 ± 194.2 324.8 ± 204.0 NS
Insulin sensitivity ((x10
-4
/min
-1
)/µU/mL)) 1.74 ± 1.0 1.55 ± 0.80 NS
Acute Insulin Response (µU/mL)
-–1
1581 ± 1074 1325 ± 795 NS
Disposition index (x10
-4
/min
-1
) 2172 ± 956 1784 ± 923 0.03
Glucose effectiveness (% per min) 0.015 ± 0.006 0.016 ± 0.009 NS

Sub-clinical measures of
atherosclerosis

CIMT (mm) 0.605 ± 0.067 0.611 ± 0.045 NS
Maximum diameter (mm) 7.55 ± 0.61 7.21 ± 0.42 0.001
Minimum diameter (mm) 6.55 ± 0.55 6.29 ± 0.40 0.006

35

Table 2-2: Unadjusted correlations between adiposity, measures of glucose
metabolism and CIMT

r-value
Adiposity Measures
Total Fat Mass 0.06
Visceral Adipose Tissue 0.06
Subcutaneous Adipose Tissue 0.03
Measures of Glucose Metabolism
Fasting Glucose
0.13
2-hr Glucose
-0.01
HbA1c
-0.04
Fasting Insulin -0.09
Insulin AUC -0.11
Insulin sensitivity 0.07
Acute Insulin Response -0.17
Disposition index -0.19
Glucose effectiveness 0.06

36

Figure 2-1: Systolic blood pressure is significantly correlated with CIMT

37

Figure 2-2: CIMT by metabolic syndrome and impaired fasting glucose status

Legend: ANCOVA used to compare means between those with and without the
metabolic syndrome or the IFG component. Covariates includes gender, age, height,
total fat and lean tissue mass.
*p=0.027

38
Table 2-3: Determinants of CIMT using multivariate regression

Total Sample

R2 Beta ± SE Standardized
Beta
p-value
Dependent variable: CIMT

Age 0.000 -0.00300 ± 0.00372 -0.093 0.376
Male gender 0.003 0.03020 ± 0.01362 0.247 0.029
Height 0.050 0.00081 ± 0.00090 0.124 0.367
HDL-cholesterol 0.051 -0.00060 ± 0.00083 -0.080 0.473
Triglycerides 0.070 -0.00019 ± 0.00012 -0.168 0.105
Waist 0.070 -0.00048 ± 0.00055 -0.102 0.391
Fasting Glucose 0.088 0.00093 ± 0.00068 0.138 0.174
2-Hour Glucose 0.091 -0.00013 ± 0.00025 -0.050 0.600
Systolic Blood Pressure 0.127 0.00162 ± 0.00076 0.270 0.035

39
CHAPTER 3: PERSISTENCE OF THE METABOLIC SYNDROME AND ITS
INFLUENCE ON CAROTID INTIMA MEDIA THICKNESS IN OVERWEIGHT LATINO
CHILDREN

Chapter 3 Abstract

The objective of this study was to examine the influence of persistence of the
metabolic syndrome and its individual components over a 3-year period on CIMT in
overweight Latino children. Ninety-seven healthy male and female overweight Latino
children (mean age at baseline: 11.0±1.8 yrs) were assessed for metabolic syndrome on
four annual evaluations and classified according to the persistence of metabolic
syndrome: NEVER (0 annual visits with the metabolic syndrome, n=53),
INTERMITTENT (1 or 2 visits with the metabolic syndrome, n=28), and PERSISTENT (3
or 4 visits with the metabolic syndrome, n=16). CIMT was measured with high-resolution
B-mode ultrasound (7.9±0.7 months after the most recent metabolic syndrome
assessment; mean age: 14.6±1.8 yr). Participants with PERSISTENT metabolic
syndrome had significantly higher CIMT (0.647±0.018 mm compared to (0.600 ±0.007
mm in those who NEVER had metabolic syndrome, p<0.01). This difference remained
significant after controlling for gender, baseline age, total fat mass and lean tissue mass,
and insulin sensitivity. PERSISTENT high waist circumference and PERSISTENT high
blood pressure were also significantly associated with higher mean CIMT, but these
differences were no longer significant after controlling for total fat and lean tissue mass.
Baseline systolic blood pressure and 2-hour glucose were significantly related to CIMT
independent of all other metabolic syndrome components (p<0.05). Persistence of the
metabolic syndrome over a 3-year period was uniquely associated with increased CIMT
during childhood. Children with hypertension, persistent abdominal adiposity and
impaired glucose tolerance may also be at higher risk for elevated CIMT.

40
Chapter 3 Introduction

In adults, a clustering of risk factors collectively known as the metabolic
syndrome has been linked to cardiovascular disease (CVD) and atherosclerosis.
51, 80

Some longitudinal studies have shown that metabolic syndrome during childhood
predicts the development of CVD by adulthood.
39, 133
The escalating obesity rates in the
pediatric population
59
and the association between obesity and metabolic syndrome in
childhood
171
substantiates the need to investigate the relationship of metabolic syndrome
and its components with atherosclerosis, especially in obese children. High-risk children
such as those who are overweight and have family history of CVD or diabetes are of
particular interest due to the concomitant occurrence of metabolic and vascular disease.
Common carotid artery intima media thickness (CIMT) has been used
predominately in adults as a non-invasive measure of atherosclerosis. CIMT is a
significant correlate to traditional vascular disease risk factors, such as cholesterol and
blood pressure
74, 130
and has been shown to be predictive of CVD in adults.
37, 78, 120
In
pediatric populations, recent cross-sectional studies have shown that obesity
136, 175, 178

and the metabolic syndrome
82
are associated with increased CIMT. Our cross-sectional
study in overweight Latino children showed there was no association between the
metabolic syndrome and CIMT.
163
No studies to date have investigated the tracking of
cardiometabolic risk factors and its effects on CIMT during childhood and adolescence.
Our current objectives are therefore: 1) to investigate the effects of persistent metabolic
syndrome over a 3 year period on a subsequent single measure of CIMT and 2) to
examine the individual effects of the persistence of each metabolic syndrome
component on a subsequent single measure of CIMT. We hypothesize that persistent
metabolic syndrome will have a significant adverse effect on CIMT.

41
Chapter 3 Methods
Subjects: Participants were enrolled in the Study of Latino Adolescents at Risk
for Diabetes (SOLAR), a longitudinal study exploring metabolic risk factors for type 2
diabetes. Study participants satisfied the following criteria for inclusion at the initial
baseline visit: 8-13 years of age, Latino ethnicity (i.e., parents and grandparents of
Latino descent), age- and gender-specific BMI " 85
th
percentile, positive family history
for type 2 diabetes, and absence of diabetes as assessed by an oral glucose tolerance
test (OGTT). Participants were excluded if they were using a medication or diagnosed
with a condition known to influence body composition or insulin / glucose metabolism.
Prior to testing procedures, written informed consent from parents and assent from the
children were obtained. This investigation was approved by the Institutional Review
Board of the University of Southern California. To be included in these analyses
participants must have 3 or 4 annual MetS assessments prior to CIMT measurement.
Study Protocol: As part of the full study protocol previously described
47
,
participants attended their annual visit at the USC General Clinical Research Center
(GCRC) for a comprehensive medical history and physical examination by a licensed
health care provider, an oral glucose tolerance test (OGTT) and body composition
measure by dual-energy x-ray absorptiometry (DEXA). Approximately 7-14 days
following the outpatient visit, participants were admitted to the USC GCRC for their
inpatient hospital visit, were examined by a licensed health care provider, and completed
an MRI. A single-slice axial TR 400/16 view of the abdomen at the level

of the umbilicus
was analyzed for cross-sectional area of visceral and subcutaneous adipose tissue.
Following an overnight fast, a frequently-sampled intravenous glucose tolerance test
(FSIVGTT) was performed the following morning.

42
Fasting lipids were assessed using Vitros Chemistry DT Slides (Johnson and
Johnson Clinical Diagnostics, Inc, Rochester, New York). Glucose was assayed using a
Yellow Springs Instruments analyzer (YSI INC., Yellow Springs, OH) that uses a
membrane bound glucose oxidase technique. Insulin was assayed using a specific
human insulin enzyme-linked immunosorbent assay kit from Linco (St. Charles, MO;
intra-assay coefficient of variation 4.7-7.0%, interassay coefficient of variation 9.1-
11.4%; cross-reaction with human proinsulin 0%).
CIMT was determined at the USC Atherosclerosis Research Unit Core Imaging
and Reading Center as previously described.
77-79, 149, 150, 176
High-resolution B-mode
ultrasound images were obtained using a Seimens Acuson CV70 (13 MHz linear array)
imager. CIMT was measured from computer-processed images of the right distal
common carotid artery approximately 1-2 cm from the bifurcation into external and
internal carotids.
We defined the metabolic syndrome using ATP-like criteria adapted for children,
as previously described by our laboratory.
47
The metabolic syndrome was determined at
each annual visit, and the subjects were then classified into 1 of 3 categories according
to the persistence of the metabolic syndrome over the repeated annual visits: NEVER
group (0 annual visits with the metabolic syndrome), INTERMITTENT group (1 or 2
annual visits with the metabolic syndrome), or PERSISTENT groups (3 or 4 annual visits
with the metabolic syndrome). The persistence of each metabolic syndrome component
was examined in the same fashion.
Statistical Analyses: Chi-square tests and analysis of variance (ANOVA) with
Bonferroni corrections were used to compare baseline characteristics of the 3 metabolic
syndrome groups. The analysis of covariance (ANCOVA) test was used to test for

43
differences in CIMT by metabolic syndrome group while adjusting for gender and the
following baseline covariates: age, total fat mass, total lean tissue mass, LDL-
cholesterol, and insulin sensitivity. To test for differences in CIMT by persistence of the
individual metabolic syndrome components, ANOVA and ANCOVA analyses were
performed to determine which component contributed to our initial result.
To determine which of the metabolic syndrome components contributed most to
the results, linear regression models were employed with the dependent variable, CIMT.
Both model approaches were adjusted for gender, age, total fat mass and total lean
tissue mass (either at baseline or at time of CIMT measurement). Data were analyzed
using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL), with an a priori
significance level set at p<0.05.
Chapter 3 Results
The total sample was composed of 57.7% male participants with a mean overall
age of 11.0 ± 1.8 yrs at baseline). In Table 3-1, baseline physical and metabolic
characteristics are shown by the three metabolic syndrome categories (NEVER,
INTERMITTENT and PERSISTENT). There were significantly more males in the
INTERMITTENT and PERSISTENT groups. The adiposity measures of BMI, total body
fat mass and abdominal adipose tissue were not statistically different across groups.
Insulin sensitivity was lowest in the PERSISTENT metabolic syndrome group, but this
difference did not reach statistical significance. Differences at baseline of the metabolic
syndrome components are shown in Table 3-2. The PERSISTENT metabolic syndrome
group had a significantly higher percentage of participants with the metabolic syndrome
at baseline and the highest mean number of metabolic syndrome components. Mean
waist circumference, systolic blood pressure and triglycerides were significantly higher in

44
the PERSISTENT metabolic syndrome group whereas mean HDL-cholesterol was
significantly lower than in the other metabolic syndrome groups (p<0.05).
Figure 3-1 shows the significantly higher CIMT with increasing persistence of the
metabolic syndrome (p=0.01), and this significance remained after adjusting for
covariates (gender, baseline age, total fat mass, total lean tissue mass and insulin
sensitivity, p<0.05). Post-hoc analyses further revealed a significantly higher mean CIMT
in the PERSISTENT than in the NEVER group (ANOVA means ± SE: 0.647 ± 0.018 mm
vs. 0.600 ± 0.007 mm, p <0.01) and a marginally significantly higher mean CIMT in
PERSISTENT than in the INTERMITTENT group (0.647 ± 0.018 mm vs. 0.611 ± 0.008
mm, p=0.09). Statistical significance remained in ANCOVA analyses.
Examination of the persistence of each metabolic syndrome component and their
individual influences on CIMT are reported in Figure 3-2. Participants with either
PERSISTENT high waist circumference (HWC) or PERSISTENT high blood pressure
(HBP) had significantly higher mean CIMT than those in the NEVER HWC or HBP
groups (p<0.05). Both models were no longer significant after adjustment for total body
fat and lean tissue mass. All other PERSISTENT component groups had higher CIMT
than the INTERMITTENT or NEVER group, but these differences did not reach statistical
significance (p>0.05).
In Table 3-3, it is shown that CIMT was significantly related with baseline SBP
(p=0.018) and impaired glucose tolerance (p=0.02), independent of gender, baseline
age, body composition and all other metabolic syndrome components. At time of CIMT
measurement, CIMT was significantly related with age (p=0.008) and total lean tissue
mass (p<0.001), but it was not associated with any of the metabolic syndrome
components.

45
Chapter 3 Discussion
The overall objective of this study was to investigate in overweight Latino youth,
the effects of the persistence of the metabolic syndrome over several years on CIMT
(measured 7.9 ± 0.7 months following the most recent metabolic syndrome evaluation).
The analysis showed that persistent metabolic syndrome over a 3-year period was
associated with a 7% higher CIMT compared to those who never had metabolic
syndrome. This finding remained significant after adjusting for a variety of covariates
including gender, baseline age, total body fat mass, total lean tissue mass and insulin
sensitivity. Of the individual metabolic syndrome components, persistent high waist
circumference and high blood pressure were related to increased CIMT, although these
effects may be explained by baseline total body fat and lean tissue mass. However,
baseline measures of systolic blood pressure and 2-hour glucose were independently
related to increased CIMT.
To date, only two longitudinal studies have focused on the relationship between
cardiovascular disease risk factors in childhood and their long-term effects on CIMT in
adulthood. The Bogalusa Heart Study found that clustering of increasing number of
metabolic syndrome components at the lower quartiles of cardiometabolic risk predicted
about a 0.100 mm decrease in CIMT at 26 year follow-up.
39
The Young Finns Study
found that adverse clustering of cardiovascular disease risk factors (LDL cholesterol,
systolic blood pressure, BMI, smoking) in childhood predicted up to 0.1 mm higher CIMT
at 21 year follow-up.
133
The present study is novel in that it shows that a significant
0.047mm greater CIMT can be observed in childhood following only 3 years of persistent
metabolic syndrome.

46
There are few studies examining the relationships between clustered
cardiometabolic risk factors and subclinical atherosclerosis during childhood. A cross-
sectional study of various pediatric metabolic syndrome definitions and CIMT found that
the definitions with higher BMI cut-offs and that utilized impaired glucose tolerance
(Viner et al. & Weiss et al.)
169, 171
were the ones with significant relationships with
CIMT.
137
The present study showed that persistent metabolic syndrome was related to
elevated CIMT, using our pediatric definition of the metabolic syndrome. This suggests
that our pediatric definition, along with Viner et al.
169
and Weiss et al.
171
, may be
suitable matches for the concept of metabolic syndrome in children. Our study also
showed that all participants in the PERSISTENT metabolic syndrome group were also
part of the PERSISTENT HWC group, which implies that abdominal adiposity may be
driving the relationship between persistent metabolic syndrome and CIMT. In contrast,
only 37.5% of those in the PERSISTENT metabolic syndrome group were also in the
PERSISTENT high blood pressure group, suggesting this feature may be more of a
stand-alone risk. Similar to Reinehr et al.
28
, we found that baseline systolic blood
pressure and 2-hour glucose were the best predictors of CIMT, independent of the other
metabolic syndrome components. These data suggest that hypertension in conjunction
with early insulin resistance and high waist circumference may be a plausible
explanation for elevated CIMT in overweight Latino youth.
The PERSISTENT group’s average CIMT was 0.047 mm higher than the NEVER
group. Studies done at the USC Atherosclerosis center have reported the normal rate of
progression reported in adult control groups ranges from 0.003 mm to 0.005 mm
increase/year.
77, 79
Therefore, this 0.047mm difference would represent about 10 years
of increased atherosclerosis progression. Although we can only speculate, such an

47
elevated CIMT may indicate an early state of diseased vessels in these adolescents,
signifying a more rapid progression of atherosclerosis as compared to youth with no
history of the metabolic syndrome. Supportive evidence includes an adult longitudinal
study that showed that every 0.030 mm increase in CIMT per year translates into a
relative risk for any coronary event of 3.1.
78
Although a direct comparison cannot be
made to our current study, a 0.047 mm CIMT difference may signify a potential coronary
risk in this high-risk youth group.
Our findings have other important clinical implications. The relatively short-term
effects of metabolic syndrome on CIMT during childhood and adolescence suggest that
more regular measurement of metabolic syndrome and more aggressive treatment for
children with the metabolic syndrome may be appropriate, particularly in high-risk groups
such as obese and family history. Waist circumference and blood pressure are easily
accessible clinic measures that would allow for cost-effective and repeatable clinical
evaluation. This could be an important predictive measure of atherosclerosis and
premature CVD events, perhaps as early as young adulthood.
The strengths of this study stem from the methodological aspects, which included the
longitudinal measures of the metabolic syndrome and a subclinical atherosclerosis
measure completed by the same sonographer and reader for the ultrasound images. In
addition, we used accurate measures of adiposity such as total and regional body
composition (DEXA and MRI scans) and direct measures of insulin sensitivity (FSIVGTT
with minimal modeling). The homogeneous sample of overweight, understudied minority
youth also contributed to the strength of this study. Despite these strengths, there were
several design limitations. The CIMT measure was only taken at a single time point and
consequently, we could only speculate that persistent metabolic syndrome caused

48
increased thickening of the carotid artery in overweight Latino youth. Repeated
measures of CIMT are currently being conducted within this cohort, but have yet to be
reported. Another limitation of the study is the unequal gender distribution, resulting in
more male participants within the PERSISTENT metabolic syndrome group. Increased
CIMT has been shown to be more prevalent in male adults
54
and male children
125
, hence
we cannot disregard male gender as a potential predictor as any gender differences may
be masked. Finally, the data results could only be generalized to overweight Latino
adolescents with a family history of type 2 diabetes.
In summary, our main conclusion is that persistence of the metabolic syndrome
over a 3-year period was associated with increased CIMT during childhood. Persistent
abdominal adiposity and/or hypertension may also have an effect on arterial structure
even before adulthood. A comprehensive evaluation for presence of the metabolic
syndrome is warranted to intervene and prevent vascular disease in this especially high-
risk group of overweight adolescents.

Table 3-1: Baseline physical and metabolic characteristics by metabolic syndrome group (n=97)

Chi-square test used for gender differences. ANOVA test was used to compare means with Bonferroni corrections for multiple
comparisons. Data are means ± SD.

NEVER
MetS
(n=53)
INTERMITTENT
MetS
(n=28)
PERSISTENT
MetS
(n=16)
p-value
Male Gender (%) 47.2% 64.3% 81.3% 0.03
Age (yrs) 10.9 ± 1.9 11.3 ± 1.6 10.6 ± 1.8 NS
Maturation stage by Tanner 2.2 ± 1.3 2.0 ± 1.1 1.7 ± 1.4 NS
Height (cm) 146.3 ± 11.6 150.3 ± 11.4 149.5 ± 14.8 NS
Weight (kg) 59.3 ± 20.2 66.8 ± 20.4 67.6 ± 21.2 NS
BMI (kg/m
2
) 26.9 ± 5.4 28.9 ± 5.9 29.4 ± 4.5 NS
Total Lean Tissue Mass (kg) 34.4 ± 9.9 37.7 ± 9.9 38.7 ± 12.0 NS
Total Fat Mass (kg) 22.7 ± 10.3 26.6 ± 11.1 26.4 ± 9.6 NS
Subcutaneous Adipose Tissue (cm
2
) 306.7 ± 158.8 344.6 ± 134.9 348.1 ± 120.7 NS
Visceral Adipose Tissue (cm
2
) 43.6 ± 20.7 50.5 ± 16.1 45.7 ± 17.9 NS

LDL-cholesterol (mg/dL) 91.9 ± 21.6 96.4 ± 25.8 96.2 ± 24.7 NS
Total cholesterol(mg/dL) 150.4 ± 26.0 151.5 ± 28.6 155.6 ± 25.9 NS
Adiponectin (µg/mL) 10.9 ± 3.1 10.2 ± 3.3 9.1 ± 2.3 NS
Fasting Glucose (mg/dl) 90.5 ± 6.3 90.3 ± 5.5 91.1 ± 5.8 NS
Fasting Insulin (!U/ml) 13.5 ± 8.2 18.0 ± 10.7 18.3 ± 12.6 NS
49

Table 3-2: Baseline descriptive statistics of the individual metabolic syndrome components by metabolic syndrome group

†p=0.06, *p<0.05, **p<0.01, ***p<0.001
Chi-square test used for data that were reported as percentages. ANOVA test was used to compare means with Bonferroni corrections
for multiple comparisons. Data are means ± SD.

NEVER
MetS
(n=53)
INTERMITTENT
MetS
(n=28)
PERSISTENT
MetS
(n=16)
p-value

Subjects with MetS (%) 0% 32.1% 87.5% 0.000
Mean Number of MetS components 1.4 ± 0.7 2.2 ± 0.9 2.9 ± 0.4 N vs. I**, N vs. P*
Waist Circumference (WC)
(cm)
85.5 ±
14.4
90.2 ± 10.9 93.1 ± 13.4 N vs. P†
Subjects meeting criteria (%) 58.5% 82.1% 100% 0.002
HDL-Cholesterol (HDL-C)
(mg/dL)
41.0 ±
10.1
34.4 ± 6.4 33.7 ± 6.9 N vs. I**, N vs. P*
Subjects meeting criteria (%) 43.4% 75.0% 81.3% 0.003
Systolic blood pressure (SBP)
(mmHg)
106.0 ±
8.4
110.3 ± 10.8 114.2 ± 8.8 N vs. P**
Subjects meeting criteria (%) 11.3% 25.0% 31.3% NS
Diastolic blood pressure (DBP)
(mmHg)
62.5 ± 6.3 63.8 ± 6.7 65.8 ± 4.6 NS
Subjects meeting criteria (%) 0% 0% 0% NS
Triglycerides (TG)
(mg/dL)
87.6 ±
43.2
103.5 ± 35.5 128.6 ± 44.8 N vs. P**
Subjects meeting criteria (%) 9.4% 21.4% 50% 0.002
2-hr Glucose
(mg/dL)
124.8 ±
16.6
123.4 ± 17.0 128.6 ± 13.4 NS
Subjects meeting criteria (%) 20.8% 14.3% 31.3% NS
50

Table 3-3: Associations between CIMT and the individual components of the metabolic syndrome at baseline and at time of
CIMT measurement

SE= Standard Error
At Baseline At time of CIMT measurement
Unstandardized
Beta
SE p-value
Unstandardized
Beta
SE p-value
Gender -0.010056 0.012073 0.407 0.023653 0.014120 0.098
Age -0.007905 0.004942 0.113 -0.010823 0.003952 0.008
Total Lean Tissue Mass 0.000000 0.000001 0.930 0.000003 0.000001 0.000
Total Fat Mass 0.000002 0.000001 0.075 0.000000 0.000001 0.509

Systolic Blood Pressure 0.001625 0.000676 0.018 0.000941 0.000827 0.259
2-hour Glucose 0.000820 0.000347 0.020 0.000146 0.000275 0.598
Waist 0.001642 0.001078 0.113 0.000428 0.001641 0.795
Triglycerides -0.000034 0.000134 0.803 -0.000052 0.000136 0.700
HDL-cholesterol -0.000008 0.000645 0.990 0.000144 0.000858 0.867

51
52

Figure 3-1: CIMT by persistence of metabolic syndrome group

Legend: MetS=metabolic syndrome.
Persistence of MetS defined as: NEVER (0 annual visits with the MetS, n=53),
INTERMITTENT (1 or 2 visits with the MetS, n=28), and PERSISTENT (3 or 4 visits with
the MetS, n=16).
53

Figure 3-2: CIMT by persistence of individual metabolic syndrome component

54

CHAPTER 4: TEMPORAL PATTERNS OF VISCERAL ADIPOSITY AND INSULIN
RESISTANCE AND THEIR EFFECTS ON CAROTID INTIMA MEDIA THICKNESS

Chapter 4 Abstract
Visceral adiposity and insulin resistance are the proposed underlying
mechanisms of the metabolic syndrome. We have previously shown that persistent
metabolic syndrome was associated with increased CIMT overweight Latino children.
Our objective was to investigate the temporal patterns of abdominal adiposity and insulin
resistance and assess if these patterns influenced CIMT. This analysis included 67
healthy male and female overweight Latino children (mean age at initial visit: 11.0±1.8
yr) from a longitudinal study of diabetes risk in children. CIMT, a measure of sub-clinical
atherosclerosis, was obtained from high-resolution B-mode ultrasound images of the
common carotid artery at one time-point. Participants were classified into tertiles of
CIMT: LOW (mean CIMT: 0.537±0.049 mm, n=22), MIDDLE (mean CIMT: 0.608±0.013
mm, n=23), and HIGH (mean CIMT: 0.667±0.032 mm, n=22). Measures of adiposity (by
DEXA and MRI), glucose metabolism (by OGTT and FSIVGTT with Minimal Model) were
completed annually over a 3-year period prior to the CIMT measurement. Repeated
measures ANCOVA was used to examine whether 3-year changes in adiposity and
insulin sensitivity influenced CIMT. Covariates included gender, baseline age, maturation
stage (as defined by Tanner) and total fat and lean tissue mass where appropriate.
There was a time*group interaction in the fasting glucose model (p<0.05), where the
HIGH CIMT group maintained constant levels of fasting glucose, whereas the LOW and
MIDDLE CIMT groups had a 3-4% decrease over time (p<0.05). All participants,
regardless of group, declined in adjusted insulin sensitivity over time (p<0.05). Changes
in insulin resistance were not associated with CIMT. Maintaining an elevated level of
fasting glucose over a 3-year period was associated with a higher CIMT.
55

Chapter 4 Introduction

The metabolic syndrome, or syndrome X as coined by Gerald Reaven, defines a
constellation of cardiometabolic features associated with the progression of diabetes and
cardiovascular disease. Proposed and generally accepted underlying mechanisms of
the metabolic syndrome include increased visceral adiposity and insulin resistance.
134, 135

In adults, the metabolic syndrome has been associated with increased cardiovascular
disease mortality.
84, 85, 117
In addition, sub-clinical states of glucose intolerance (or pre-
diabetes) have also been associated with 15-54% greater cardiovascular disease
mortality.
146
Hence, the sequence of events leading to the onset of cardiovascular
disease events (including atherosclerosis) have been linked to insulin resistance.
70
In
fact, the increased risk for cardiovascular disease has even been shown to appear prior
to the onset of type 2 diabetes in adult populations
97
, including Latinos.
71, 104

To date, three cross-sectional studies in obese children have shown that insulin
resistance is an independent risk factor for increased CIMT.
13, 61, 137
A longitudinal report
from our laboratory has shown that children with either persistent metabolic syndrome or
persistent pre-diabetes have progressively increasing adiposity, decreasing insulin
sensitivity and beta cell function over time.
66, 167
In another study, we also showed that
children with persistent metabolic syndrome had higher CIMT than those who never had
the metabolic syndrome.
162
These data support abdominal adiposity and insulin
resistance as a potential predictors of subclinical atherosclerosis. The purpose of this
study is to examine the influence of temporally-related changes in adiposity and insulin
action on CIMT. We hypothesized that children with declining insulin sensitivity and
increasing visceral adiposity would have a higher CIMT than those with stable metabolic
profiles.
56

Chapter 4 Methods

Subjects: Participants were enrolled in the Study of Latino Adolescents at Risk
for Diabetes, a longitudinal study exploring metabolic risk factors for type 2 diabetes.
Study participants satisfied the following criteria for inclusion at the initial baseline visit:
8-13 years of age, Latino ethnicity (i.e., parents and grandparents of Latino descent),
age- and gender-specific BMI !85
th
percentile, positive family history for type 2 diabetes,
and absence of diabetes as assessed by an oral glucose tolerance test. Participants
were excluded if they were using a medication or diagnosed with a condition known to
influence body composition or insulin / glucose metabolism. Prior to testing procedures,
written informed consent from parents and assent from the children were obtained. This
investigation was approved by the Institutional Review Board of the University of
Southern California.
Study Protocol: As part of the full study protocol previously described
47
,
participants attended their annual visit to at the USC General Clinical Research Center
for a comprehensive medical history and physical examination by a licensed health care
provider, an oral glucose tolerance test and body composition measure by dual-energy
x-ray absorptiometry. Approximately 7-14 days following the outpatient visit, participants
were admitted to the GCRC for their inpatient hospital visit and examined once more by
a licensed health care provider and an MRI was completed. A single-slice axial TR
400/16 view of the abdomen at the level

of the umbilicus was analyzed for cross-
sectional area of visceral and subcutaneous adipose tissue. Following an overnight fast,
a frequently-sampled intravenous glucose tolerance test was performed the following
morning.
57

Fasting lipids were assessed using Vitros Chemistry DT Slides (Johnson and
Johnson Clinical Diagnostics, Inc, Rochester, New York). Glucose was assayed using a
Yellow Springs Instruments analyzer (YSI INC., Yellow Springs, OH) that uses a
membrane bound glucose oxidase technique. Insulin was assayed using a specific
human insulin enzyme-linked immunosorbent assay kit from Linco (St. Charles, MO;
intra-assay coefficient of variation 4.7-7.0%, interassay coefficient of variation 9.1-
11.4%; cross-reaction with human proinsulin 0%).
CIMT was determined at the USC Atherosclerosis Research Unit Core Imaging
and Reading Center as previously described.
77-79, 149, 150, 176
High-resolution B-mode
ultrasound images were obtained using a Seimens Acuson CV70 (13 MHz linear array)
imager. CIMT was measured from computer-processed images of the right distal
common carotid artery approximately 1-2 cm from the bifurcation into external and
internal carotids.
Statistical Analyses: Sixty-seven participants were classified in 3 tertile groups of
the outcome measure CIMT: LOW (mean CIMT: 0.537±0.049mm, n=22), MIDDLE
(mean CIMT: 0.608±0.013mm, n=23), and HIGH (mean CIMT: 0.667±0.032mm, n=22).
Baseline characteristics of the 3 groups were compared using chi-square tests and
ANOVA with Bonferroni corrections. Three-year changes in adiposity as well as glucose
metabolism, measured by insulin sensitivity, acute insulin response and disposition
index, were analyzed with repeated measures ANCOVA. For ANCOVA, the following
covariates were included in all models: gender, baseline age and Tanner stage. Baseline
total lean tissue mass was used as a covariate when total body fat mass was examined
as an outcome. In the visceral adiposity models, baseline subcutaneous adiposity was
entered as a covariate. Baseline total fat mass and total lean tissue mass were used as
58

covariates for models containing insulin sensitivity, acute insulin response and
disposition index as outcomes. For repeated measures ANCOVA, Mauchly’s test of
sphericity was used to assess the form of the common covariance matrix. When the
sphericity assumption was not met, the Huynh-Feldt correction was used. Data were
analyzed with SPSS version 13.0 (SPSS Inc, Chicago, IL), and type 1 error was set at
"<0.05.
Chapter 4 Results

Baseline unadjusted descriptive characteristics of the participants by CIMT tertile
group are shown in Table 4-1. The sample was composed of 58.4% males and there
were no significant gender, age or Tanner differences by group. Height, weight, BMI,
body composition and abdominal adiposity were also not statistically different by tertile
group. Baseline unadjusted glucose and insulin metabolism indices and the metabolic
syndrome components of the three CIMT tertile groups are depicted in Table 4-2. There
were no significant baseline differences in glucose and insulin metabolism measures or
most of the metabolic syndrome components by CIMT tertile group. There were
longitudinal differences in adiposity measures by CIMT tertile group. Although overall
BMI and BMI percentile did not change significantly (data not shown), there was an
interaction for time*group for total fat mass (p=0.07). There was a time*group interaction
in the visceral adiposity model (p<0.05), such that the HIGH CIMT group had a 26%
increase compared to a 3% decrease in the MIDDLE CIMT group (p<0.05).
There were no longitudinal differences in insulin action measures (data not
shown). All participants, regardless of CIMT tertile group, declined in adjusted insulin
sensitivity over time (p<0.05). There was a time*group interaction in the fasting glucose
model (Figure 4-3, p<0.05), where the LOW and MIDDLE CIMT groups had a 3-4%
59

decrease compared to HIGH CIMT group that maintained constant levels of fasting
glucose (p<0.05).
Chapter 4 Discussion
This study examined temporal changes of various cardiometabolic risk factors on
CIMT in an overweight Latino adolescent cohort. We hypothesized an association
between visceral adiposity and insulin resistance with CIMT. A significant interaction was
observed with visceral adiposity although it was not easily interpretable. Insulin
sensitivity declined in all participants over the 3-year period; there was no clear
relationship between any measure of insulin resistance and CIMT. Children in the
highest CIMT tertile maintained constant levels of fasting glucose whereas the children
in the lower tertiles of CIMT had a 3-4% decrease in fasting glucose.
Insulin resistance and increased visceral adiposity along with the metabolic
syndrome have been associated with atherosclerosis and vascular disease in adults. In
the pediatric literature, CIMT has been related to insulin resistance
13, 61
and impaired
glucose tolerance.
137
The results of our study did not concur with the literature in
children. We must not dismiss the possibility that the mechanisms implicated in insulin
resistance and development of subclinical atherosclerosis may not necessarily apply to
our study due to pubertal growth change and patterns. Pubertal changes in children and
adolescents affect insulin resistance, body fat distribution and complex hormonal
changes.
90
In fact, in the SOLAR study we showed that in response to declining insulin
sensitivity, pre-pubertal children have a compensatory acute insulin response whereas
children in puberty or post-puberty do not.
67
In addition, we studied a highly specific
cohort of overweight and highly insulin resistant Latino children with insulin sensitivity
levels below 3.0 x10
-4
min
-1
/uU/ml. Having such a narrow range of insulin sensitivity may
have hindered any potential relationships with CIMT.
60

Fasting glucose levels in children as a risk factor for subclinical atherosclerosis
and future cardiovascular disease must not be disregarded. A large meta-analysis of 20
studies in over 90,000 adults showed that high fasting glucose is a risk factor for
cardiovascular disease
44
and recommendations for glycemic control as a preventive
measure has been recently asserted for youth with type 2 diabetes.
151
Despite this
support and recommendation for fasting glucose as a marker for atherosclerosis risk,
other studies have shown that glucose control is not associated with improved
cardiovascular outcomes.
91, 160
Moreover, the literature in pediatric populations is sparse.
A previous unpublished study from our laboratory (data shown in Chapter 2) shows that
in a cross-sectional analysis, impaired fasting glucose status is associated with
increased CIMT. These results are supported by Reinehr et al.’s report
137
of pertinent
metabolic correlates of CIMT in overweight youth, which showed that impaired fasting
glucose is a predictor of elevated CIMT in children. In the present study, fasting glucose
was assessed over a 3-year period and those children in the highest CIMT tertile
maintained a mean fasting glucose level above 90mg/dL, compared to those in the lower
CIMT tertiles who had a 4% decrease in glucose levels over a 2-year period. Despite
the normal glucose levels of participants in the high CIMT tertile group, atherosclerosis
risk, like type 2 diabetes risk, is on a continuum; therefore defining a clear cut-off would
be ill-considered. Based on our findings it may also be imprudent to conclude that
glucose control in children is protective of subclinical atherosclerosis risk, however our
findings warrant future research, particularly in high-risk populations.
The strengths of this study include the use of subclinical atherosclerosis
measures with a single sonographer and reader for the ultrasound images. In addition,
accurate measures of total and regional body composition (DEXA and MRI scans) and
direct measures of insulin sensitivity (FSIVGTT with minimal modeling) were utilized.
61

The large homogeneous sample of understudied minority youth also contributed to the
strengths of this report. Several limitations are also noteworthy. There were no lean
participants to use as matched controls and as a consequence, this may have severely
limited the findings. Finally, the data results can only be generalized to the very distinct
group of overweight Latino adolescents with a family history of type 2 diabetes.
In conclusion, children in the highest CIMT tertile maintained constant levels of
fasting glucose whereas the children in the lower tertiles of CIMT had a 3-4% decrease
in fasting glucose. Overweight Latino adolescents with decreasing insulin sensitivity and
uncontrolled fasting glucose levels, may be at higher risk for subclinical atherosclerosis.
Table 4-1: Baseline unadjusted descriptive characteristics by tertiles of CIMT
Variables

CIMT Categories
Low CIMT
Tertile
(n=22)
Middle CIMT
Tertile
(n=23)
High CIMT
Tertile
(n=22)
Gender (Male) 24 19 23
Age (years) 11.7 ± 1.8 11.7 ± 1.8 11.6 ± 1.6
Tanner stage (median) 1 1 2
Height (cm) 151.6 ± 9.8 150.8 ± 11.6 153.7 ± 11.7
Weight (kg) 66.6 ± 18.9 67.3 ± 19.2 71.2 ± 21.5
BMI (kg/m
2
) 28.5 ± 5.5 29.0 ± 4.7 29.5 ± 5.6
BMI percentile 96.8 ± 3.5 97.7 ± 2.5 97.1 ± 4.1
Total Lean Tissue Mass
(kg)
38.9 ± 10.1 37.8 ± 9.7 41.0 ± 12.3
Total Fat Mass (kg) 24.9 ± 9.5 26.4 ± 9.5 27.8 ± 10.1
SAAT (cm
2
) 345.0 ± 155.0 347.3 ± 132.6 374.0 ± 154.9
VAT (cm
2
) 41.4 ± 17.1 47.3 ± 21.7 48.5 ± 21.5
ANOVA performed to compare means with Bonferroni corrections for multiple comparisons and
data are means ± standard deviations. Chi square was used for gender and Tanner comparison.
62

Table 4-2: Unadjusted baseline glucose metabolism and insulin action measures
by CIMT tertiles
Variables

CIMT Categories
Low CIMT
Tertile
(n=22)
Middle CIMT
Tertile
(n=23)
High CIMT
Tertile
(n=22)
Fasting glucose (mg/dl) 89.9 ± 5.9 91.3 ± 9.5 90.0 ± 6.9
2-hour glucose (mg/dl) 122.8 ± 21.6 124.4 ± 15.7 128.4 ± 15.3
Glucose IAUC (mg/min/dl) 265.5 ± 39.7 257.4 ± 28.9 266.7 ± 29.8
Fasting insulin (!U/ml) 16.3 ± 8.8 17.7 ± 10.6 16.3 ± 11.0
2-hour insulin (!U/ml) 164.6 ± 132.9 171.3 ± 118.2 201.1 ± 181.6
Insulin IAUC (!U/min/ml) 289.4 ± 162.6 299.8 ± 175.3 298.4 ± 172.5
SI (x10
-4
min
-1
/!U/ml) 1.93 ± 1.14 2.06 ± 1.54 1.70 ± 0.96
AIR (!U/ml x10 min) 1660 ± 1130 1564 ± 894 1810 ± 1153
DI (x10
-4
min
-1
) 2487 ± 1067 2494 ± 1360 2369 ± 1063
ANOVA performed to compare means with Bonferroni corrections for multiple comparisons and
data are means ± standard deviations.
63

Figure 4-1: Insulin sensitivity declined in all CIMT tertiles

Legend: Repeated measures ANOVA used to compare changes in insulin sensitivity
over a 3-year period by CIMT tertiles. Data are means ± standard error.
All analyses are adjusted for gender and baseline age, and Tanner stage. Sphericity
assumption was violated and Huynh-feldt correction used.
64

Figure 4-2: Temporal patterns of visceral adiposity by CIMT tertiles

Legend: Repeated measures ANOVA used to compare changes in visceral adiposity
over a 3-year period by CIMT tertiles.
Data are means ± standard error.
All analyses are adjusted for gender and baseline age, and Tanner stage. Sphericity
assumption was violated and Huynh-feldt correction used.

65

Figure 4-3: Distinct temporal patterns for fasting glucose by CIMT tertiles

Legend: Repeated measures ANOVA used to compare changes in fasting glucose over
a 3-year period by CIMT tertiles. Data are means ± standard error.
All analyses are adjusted for gender and baseline age, and Tanner stage. Sphericity
assumption was violated and Huynh-feldt correction used.

66

CHAPTER 5: PROGRESSION OF CAROTID INTIMA MEDIA THICKNESS AND ITS
RELATIONSHIP TO CARDIOMETABOLIC RISK FACTORS

Chapter 5 Abstract
The objective was to assess CIMT change over 2 years in overweight Latino
adolescents and examine its relationship to cardiometabolic risk. 72 healthy overweight
male and female Latino adolescents (mean age: 14.5±1.7 yrs; mean BMI: 31.5±6.9
kg/m
2
) were evaluated at baseline and 2 years later for: CIMT by high resolution B-
mode ultrasound, the metabolic syndrome and its features, body composition by DEXA
and MRI, and glucose/insulin measures by fasting blood, and oral and intravenous
glucose tolerance tests. CIMT at baseline did not differ from 2-year follow-up; however
38 participants increased CIMT (Mean progression ± SD: 0.017 ± 0.003 mm; +2.8%)
and 34 decreased (Mean regression ± SD: -0.019 ± 0.002 mm; -3.1%). ANCOVA
analyses show that participants with CIMT progression had higher baseline LDL-
cholesterol and total cholesterol (91.3 ± 3.4 and 150.3 ± 3.9 mg/dL) compared to those
with CIMT regression (78.1 ± 3.6 and 135.6 ± 4.2 mg/dL, p<0.05), independent of
gender, baseline CIMT, age, and height. Adiposity and other measures of insulin action
had no significant relationships with CIMT change. In multivariate regression, LDL-
cholesterol was the sole predictor of CIMT progression, but the effect was small (odds
of CIMT progression increased by 3% for each 1 mg/dL higher baseline LDL-cholesterol
[95% confidence interval (CI), 1.004-1.006, p=0.03]. These results indicate a high
variability in the magnitude of CIMT change in growing overweight Latino youth.
Adiposity and insulin resistance were not associated with CIMT change and LDL-
cholesterol was the sole predictor of CIMT progression. These results support the use
of LDL-cholesterol to assess sub-clinical atherosclerosis risk in overweight Latino youth.
67

Chapter 5 Introduction
The increasing prevalence of pediatric childhood obesity
59
warrants investigation
into the link between obesity and atherosclerosis risk in youth. The Latino population in
the US is rapidly growing and has shown a variety of obesity related co-morbidities,
such as hypertension and diabetes, placing them at high-risk for cardiovascular
disorders. Latino children have a high prevalence of obesity
59
, insulin resistance
65
,
metabolic syndrome
47
and impaired fasting glucose/pre-diabetes
138
, all of which may
contribute to the early development of atherosclerosis.
Carotid artery intima media thickness (CIMT) is a noninvasive measure of
subclinical atherosclerosis. Increased CIMT has been shown to start in youth and its
correlates are similar to those in adulthood. Yet, the progression of CIMT in youth has
not been widely examined. Increased thickness of the carotid artery and its associated
cardiovascular disease events in adulthood has various inter-related predictors such as
obesity, male gender, metabolic dysfunction, dyslipidemias and hypertension. Studies
over the past decade have shown similar relationships of elevated CIMT and traditional
cardiometabolic risk factors in children and young adults. For instance, cross-sectional
studies have shown that overweight children had higher CIMT compared to their normal
weight counterparts.
136, 175, 178
Boys have also been shown to have higher CIMT than
girls.
30, 126, 172
CIMT in children has also been associated with glucose and/or insulin
metabolism dysfunction
13, 61, 137
, elevated cholesterol levels
114, 172
and high blood
pressure.
30, 102, 156
To date, our laboratory has completed the only study that includes
repeated measures of various cardiometabolic risk factors in overweight Latino youth.
From these data we have previously shown that children with persistent metabolic
syndrome over a 3-year period exhibited a higher CIMT than those who never had the
metabolic syndrome.
162
Of the metabolic syndrome components, high blood pressure
68

and high waist circumference were the features most highly associated with higher
CIMT.
162
To follow-up on these findings, we now present longitudinal measures of CIMT
in overweight Latino youth.
The purpose of this paper was to assess the longitudinal change in CIMT and
the association of CIMT progression to various cardiovascular risk factors at baseline.
The first objective of this study was to assess the change of CIMT over a 2-year period.
Next, we assessed the relationship of CIMT progression to potential CIMT predictors,
including adiposity, the metabolic syndrome and its individual features, lipids and insulin
resistance. Finally, we examined any potential gender differences in these associations.
Based on the literature and our prior published results, we hypothesized that baseline
systolic blood pressure, abdominal adiposity and insulin resistance would predict CIMT
progression.
Chapter 5 Methods
Subjects: Participants were enrolled in the Study of Latino Adolescents at Risk
for Diabetes (SOLAR), a longitudinal study exploring metabolic risk factors for type 2
diabetes. Study participants satisfied the following criteria for inclusion at the initial
baseline visit: 8-13 years of age, Latino ethnicity (i.e., parents and grandparents of
Latino descent), age- and gender-specific BMI ! 85
th
percentile, positive family history
for type 2 diabetes, and absence of diabetes as assessed by an oral glucose tolerance
test (OGTT). Participants were excluded if they were using a medication or diagnosed
with a condition known to influence body composition or insulin / glucose metabolism.
Prior to testing procedures, written informed consent from parents and assent from the
children were obtained. This investigation was approved by the Institutional Review
Board of the University of Southern California. To be included in this analysis, SOLAR
69

participants must have had a baseline CIMT in 2006 (n=123) and a repeat CIMT
measure in 2008 (n=73) and all measures of cardiometabolic risk. One participant was
diagnosed with diabetes and was therefore excluded from this analysis. The final
sample was composed of 72 participants.
Study Protocol: Details of the longitudinal study protocol and design have been
previously described.
47
In brief, participants attended two annual visits at the USC
General Clinical Research Center. On the first visit, participants received a
comprehensive medical history and physical examination by a licensed health care
provider. Clinical staff then collected vital signs, blood pressure in triplicate and
performed a 2-hour oral glucose tolerance test (OGTT). Approximately 7-14 days
following the outpatient visit, participants were admitted for an inpatient visit at the USC
GCRC for their second visit. They were examined by a licensed health care provider
and were given dinner. Following a supervised, overnight fast, a 3-hour modified
frequently-sampled intravenous glucose tolerance test (FSIVGTT, with 13 time points)
was performed by certified nursing and phlebotomy staff.
Body composition measures and CIMT measure were performed at either visit
based on availability of the participant and staff. Total body composition was completed
by dual-energy x-ray absorptiometry and magnetic resonance imaging was used to
assess abdominal adiposity. Using a GE 1.5 Signa LX-Ecospeed with 1.5 Tesla
magnet (GE Healthcare, Piscataway, New Jersery), a single-slice axial TR 400/16 view
of the abdomen at the level

of the umbilicus was analyzed for cross-sectional area of
visceral and subcutaneous abdominal adipose tissue. CIMT was determined at the USC
Atherosclerosis Research Unit Core Imaging and Reading Center as previously
described.
77, 78, 149, 150, 176
High-resolution B-mode ultrasound images were obtained
70

using a Seimens Acuson CV70 (13 MHz linear array) imager. C-IMT was measured
from computer-processed images of the right distal common carotid artery
approximately 1-2 cm from the bifurcation into external and internal carotids.
Fasting blood draws from the 1
st
and 2
nd
visits were collected to assay for fasting
lipids (triglycerides, total, LDL- and HDL-cholesterol), glucose and insulin. Lipids were
assessed using Vitros Chemistry DT Slides (Johnson and Johnson Clinical Diagnostics,
Inc, Rochester, New York). Glucose was assayed using a Yellow Springs Instruments
analyzer (YSI INC., Yellow Springs, OH) that uses a membrane bound glucose oxidase
technique. Insulin was assayed using a specific human insulin enzyme-linked
immunosorbent assay kit from Linco (St. Charles, MO; intra-assay coefficient of
variation 4.7-7.0%, interassay coefficient of variation 9.1-11.4%; cross-reaction with
human proinsulin 0%).
Glucose and insulin area under the curve were calculated from the OGTT data
(in mg/dL for glucose and #U/min/mL for insulin) as six time points. To determine insulin
sensitivity and acute insulin response, values for glucose and insulin from the modified
FSIVGTT were entered into MinMod Millennium 2003, version 5.16 (Richard Bergman,
University of Southern California, Los Angeles, CA). The disposition index, an index of
the compensatory adaption to insulin resistance, was calculated as the product of
insulin sensitivity and acute insulin response and used to approximate beta cell
function. Glucose effectiveness, also derived from the minimal model, is the ability of
glucose to suppress endogenous glucose production and stimulate glucose uptake,
independent of an increment of insulin
24
.
71

Metabolic Syndrome Assessment: Using a definition similar to the Adult
Treatment Panel
2
that we adapted for pediatric populations, the metabolic syndrome
was defined by the presence of at

least three of the following components:
• abdominal obesity (waist circumference !90th percentile for age, gender and
Hispanic ethnicity)
56
,
• hypertriglyceridemia (triglycerides !90th percentile for age and gender)
75
,
• low HDL

cholesterol (HDL cholesterol $10th percentile for age and gender)
75
,
• high blood pressure (either systolic or diastolic blood pressure >90th

percentile
adjusted for height, age, and gender)
6
,
• impaired glucose tolerance (OGTT 2-hour glucose: 140 -199 mg/dl)
4
.

Statistical Procedures: Each participant’s CIMT change was calculated by taking
the difference of the 2-year follow-up CIMT from the baseline CIMT. In preliminary
analyses, CIMT change was used as a continuous variable while in ANCOVA analyses,
participants were placed in one of 3 groups: Non-progression (CIMT change $0.000mm,
n=34), Normal Progression (CIMT change >0.000mm and <0.01mm, n=15) or
Advanced Progression (CIMT !0.01mm, n=23). Advanced CIMT Progression was
based on a conservative cut-off using normal rates of CIMT progression in healthy
adults (approximately 0.005 mm/year hence, !0.01mm over 2 years was deemed
advanced progression). For logistic regression, CIMT progression was defined to
include any CIMT progression (CIMT !0.01mm) or Advanced CIMT progression only
(CIMT !0.01mm). The independent variables (adiposity measures, metabolic
syndrome, blood pressure, lipids and measures of glucose/insulin metabolism) were
measured at the baseline and follow-up CIMT assessments. The Shapiro-Wilkes W
72

test was used to test the Gaussian distribution of residual values of all continuous
variables, none of which deviated from normality (p>0.05).
For descriptive analysis, independent t-test with Levene’s test for equality of
variances and chi-square tests were performed to assess mean physical and metabolic
characteristics by gender at baseline and 2-year follow-up. This was followed by a
preliminary analysis of simple and partial correlations of CIMT change with each
baseline independent variable. This analysis was repeated with CIMT change and
changes in adiposity (BMI, BMI z-score, total fat mass, visceral and subcutaneous
adiposity), blood pressure, lipids (triglycerides, LDL-, HDL- and total cholesterol), and
glucose and insulin indices (fasting and 2-hour glucose and insulin, insulin sensitivity,
acute insulin response, disposition index, glucose and insulin AUC). Next, ANCOVA
was used to compare CIMT progression groups and their baseline and change of all the
cardiometabolic risk factors previously mentioned. A priori covariates included gender
and baseline age and height. These covariates were used in adjusted analyses due to
their documented effects on CIMT progression. The baseline measure was always
used to adjust for any analysis of 2-year change in the variables of interest.
Significant associations were further examined by multivariate linear regression
and by 2-way ANCOVA for the metabolic syndrome. Interaction terms were used to test
whether gender significantly modified the relationship between each of the
cardiovascular risk factors and change in CIMT, while adjusting for baseline CIMT, age
and height.
Multiple logistic regression analysis was employed to determine the relative
contribution of the baseline predictors (independent continuous variables in model 1:
fasting insulin, glucose effectiveness and LDL-cholesterol) to the CIMT change (coded
73

as a binary dependent variable: CIMT non-progression and CIMT progression), while
considering the confounding effects of gender, baseline age and height. The second
model included the same independent variables but used CIMT coded as a binary
variable with CIMT non-progression and Advanced CIMT progression. Data were
analyzed using SPSS for Mac version 16.0 (SPSS Inc., Chicago, IL), with an a priori
significance level of p<0.05.
Chapter 5 Results
Descriptive statistics of baseline physical and metabolic characteristics of 72
Latino adolescents (38/34 Male/Female; mean age: 14.5 ± 1.5 years) are shown in
Table 5-1. Tanner stage for males ranged from 1-5, whereas in females the range was
limited to the later pubertal stages 3-5, yet the gender disparity in pubertal stages was
not significantly different (p>0.05). Males were significantly taller and had higher total
lean tissue mass than females (p<0.001). Systolic and diastolic blood pressures were
higher in males than females (p<0.05). HDL-cholesterol was significantly lower in
males than in females (p<0.05). Females had a lower disposition index than males
(p<0.05). CIMT did not differ by gender but females had a lower maximum carotid
diameter than males (p<0.05). Body composition or cardiometabolic risk characteristics
did not significantly differ between baseline and 2-year follow-up (data not shown), with
the exception of fasting glucose that was higher in at the baseline measure (mean ±
SD: 88.3 ± 7.6 vs. 85.8 ± 8.0 mg/dL at follow-up, p<0.05).
Of the 72 participants, 38 showed an increase in CIMT (mean increase: 0.017 ±
0.003 mm, +2.8%) and 34 participants showed a decrease in CIMT (mean decrease ±
SE: -0.019 ± 0.002 mm, -3.1%). In the whole sample, mean CIMT at baseline (0.605 ±
0.059 mm) and follow-up 0.605 ± 0.055 mm) were not significantly different (p>0.05).
74

Simple and partial correlations between baseline cardiovascular risk factors and
CIMT change revealed that LDL-cholesterol, fasting insulin and insulin AUC were
positively correlated to change in CIMT (r=0.21 – 0.24, p<0.05) with glucose
effectiveness the only factor negatively correlated with change in CIMT (r= -0.30,
p=0.01). These relationships were no longer significant after adjusting for gender and
baseline CIMT, age and height, except for the inverse relationship with glucose
effectiveness and CIMT change, which remained significant (Figure 5-1, r= -0.30,
p=0.01). Correlations between baseline body composition (including abdominal
adiposity), insulin sensitivity and blood pressure with CIMT change were not significant
(data not shown). Correlations between change in any given cardiometabolic risk factor
and CIMT change were also not significant (p>0.05) data not shown). There were no
significant gender interactions independent of baseline CIMT (p>0.05, data not shown).
Participants with the metabolic syndrome at baseline showed an increase in
CIMT change compared to those who did not have the metabolic syndrome, but this
difference did not reach significance (ANOVA, 0.007 ± 0.005 vs. -0.003 ± 0.003mm,
p=0.16). Gender did not modify the effect of CIMT change between participants with or
without the metabolic syndrome (2-way ANOVA interaction, p>0.05). In Figure 5-2,
baseline LDL- and total cholesterol were significantly higher in the Advanced CIMT
Progression Group (92.6 ± 4.4 and 152.9 ± 5.1 mg/dL) versus those in the CIMT Non-
progression group (77.7 ± 3.7 and 135.5 ± 4.2 mg/dL, p<0.05), independent of gender,
baseline CIMT, age, height and HDL-cholesterol. All other cardiometabolic risk factors
had no association with the 3 CIMT progression groups (p>0.05, data not shown).
Two logistic regression models (Table 5-2) were used to determine the
independent predictors of CIMT progression, which was evident in 53% of the sample.
In model 1, LDL-cholesterol was the sole predictor of any CIMT progression
75

(CIMT>0.000mm), where the odds for CIMT progression over a 2-year period was 1.03
for each 1 mg/dL higher baseline LDL-cholesterol [95% confidence interval (CI, 1.004-
1.006, p=0.03]. Model 2 shows that LDL-cholesterol was a marginally significant
predictor of advanced CIMT progression.
Chapter 5 Discussion
This study is the first to examine CIMT progression during childhood growth and
development and its cardiometabolic predictors in healthy, overweight Latino youth. Our
results indicate that change in CIMT was highly variable; 53% of the participants
exhibited an increase in CIMT while the remaining 47% of participants showed a
decrease in CIMT. Moreover, 36% of participants showed progression beyond that of
the physiological norm (Advanced CIMT Progression Group with CIMT !0.01mm over 2
years). Participants in the advanced CIMT progression group had significantly higher
baseline LDL and total cholesterol than those in the CIMT non-progression group.
Another predictor of CIMT change was baseline glucose effectiveness, which had a
negative relationship with CIMT change independent of gender and baseline CIMT, age
and height. Finally, the odds of CIMT progression increased 1.03 times (or 3%) for
each 1mg/dL increase of baseline LDL-cholesterol, independent of glucose
effectiveness and other covariates. These results highlight the potential effects of LDL-
cholesterol in youth on advanced CIMT progression.
Contrary to our hypothesis, baseline systolic blood pressure, abdominal
adiposity and insulin sensitivity were not associated with change in CIMT over a 2-year
period. We have previously shown that Latino children with persistent high blood
pressure and high waist circumference, along with individuals with persistent metabolic
syndrome have a higher CIMT. Our present report shows that these same risk factors
76

were not associated with the rate of CIMT progression. Instead, LDL-cholesterol was
the defining cardiometabolic risk factor that may have elicited more CIMT progression.
These findings supplement our previous predictors of persistent metabolic syndrome,
high blood pressure and high waist circumference as the predictors of elevated CIMT.
This was not entirely surprising since the cardiometabolic risk factors that were
predictive of a single measure of elevated CIMT may not necessarily be the same as
those that predicate a faster rate of CIMT progression. It suggests that multiple risk
factors are involved in both elevated CIMT and CIMT progression.
The relationship between LDL-cholesterol and CIMT has been strongly
documented in adults but is still ambiguous in studies of healthy children; 2 studies
have observed this relationship
164, 170
whereas 2 others have not.
99, 128
We have shown
a statistically significant relationship with LDL-cholesterol and CIMT progression after
only 2 years in overweight Latino youth with a family history of type 2 diabetes. Our data
showed that small differences in LDL-cholesterol at baseline were still sufficient to
observe differences between CIMT progressors and non-progressors. Further analyses
of our data using repeated measures ANCOVA revealed that participants with
advanced CIMT progression maintained significantly higher LDL cholesterol over the 2-
year period (93.5±4.4 to 90.1±5.4 mg/dL) than those who did not show CIMT
progression (77.2±3.8 to 82.8±4.6 mg/dL, p=0.02) after controlling for gender and
baseline age and HDL-cholesterol. This supplements our findings by showing that
maintaining higher LDL-cholesterol levels could add to CIMT progression beyond that of
the physiological norm. It is also important to note that only 5 participants (all male) had
a clinically abnormal level of LDL-cholesterol level (above the 90
th
percentile for gender
and age).
75
We cannot rule out the possibility that these participants may have family
histories of hypercholesterolemia. Studies have shown that children with familial
77

hypercholesterolemia consistently have increased CIMT compared to healthy
controls.
86, 99, 128, 164, 170
However, we cannot attest that a family history of
hypercholesterolemia contributed to our findings due to a lack of thorough
cardiovascular disease family history data.
Dysfunction of glucose and insulin metabolism has been associated with
atherosclerosis and vascular disease in adults. Studies in children show that fasting
glucose and/or insulin and insulin resistance measures derived from a single measure
(such as HOMA-IR, QUICKI) have been associated with CIMT.
13, 18, 61, 137
Only one
study has assessed insulin resistance using an OGTT and it showed no significant
relationship between insulin resistance and CIMT.
13
Similarly, we found no significant
associations between insulin resistance (using the FSIVGTT and minimal modeling)
and CIMT change. While we did observe some interesting relationships with fasting
insulin and insulin area under the curve, these associations were not independent of the
baseline CIMT measure. This may suggest that measuring insulin levels, separate from
insulin resistance indices, in conjunction with a CIMT measure may provide some
insight to CIMT progression. A rather unexpected finding was the negative relationship
between glucose effectiveness and CIMT change. Glucose effectiveness is derived
from the FSIVGTT data with minimal modeling and is defined as the ability of glucose to
both suppress endogenous glucose production and stimulate glucose uptake.
31
If lower
baseline glucose effectiveness leads to a greater CIMT progression over a 2-year
period, we can speculate that this disruption of glucose homeostasis may be the early
stage of a physiological mechanism (such as oxidative stress) that eventually leads to
arterial wall dysfunction. Interestingly, in a separate longitudinal analysis of this same
cohort (unpublished), we reported that those with impaired fasting glucose over time
(!100mg/dL) had significantly higher CIMT. We were unable to conduct a similar
78

analysis on this sub-sample due to an extremely low number of participants with
impaired fasting glucose. Finally, although no other relationships between any other
glucose measurements and CIMT change were found, there was a significant negative
correlation between baseline glucose effectiveness with fasting glucose (p<0.05).
A critical clinical implication arises from our findings and it is to provide support
for a pediatric risk assessment for sub-clinical atherosclerosis. Currently, there are no
screening guidelines for subclinical atherosclerosis in high-risk youth. The Primary
Prevention Writing Group III for the American Heart Association (AHA) Prevention
Conference V emphasized the potential value of risk assessment when CIMT measures
were used in conjunction with traditional risk factor assessments.
68
Given the growing
literature on the early development of atherosclerosis in children, along with added
predispositions to metabolic disorders, there is little reason not to extend this
recommendation to children with these health disadvantages. Our results suggest that a
simple fasting blood draw to assess insulin and lipids, plus a non-invasive CIMT
measure would be potential candidates to assess risk. Once a pediatric risk
assessment is instituted, proper treatment indications would follow including diet and
exercise prescriptions. A few studies have reported the benefits of diet and exercise
interventions in youth for the purpose of improving vascular function and arterial
thickness.
55, 83, 108
More studies should address the impact and efficacy of establishing
these clinical guidelines and treatments in high-risk youth.
The strengths of this study include its longitudinal measures of subclinical
atherosclerosis using the same sonographer and reader of the ultrasound images for all
baseline and follow-up CIMT measures. Assessment of cardiovascular risk was done
with clinical measures of total and regional body composition (DEXA and MRI scans)
79

and direct measures of insulin sensitivity (FSIVGTT with minimal modeling). In addition,
simple and clinically applicable measures of fasting blood were employed. The use of a
large homogeneous sample of understudied minority youth contributes to the strengths
of this report, but also limits the generalizability of the results to overweight Latino
adolescents with a family history of type 2 diabetes. The short time frame and relatively
small sample of this longitudinal study may have impeded identification of correlates of
CIMT progression. Future CIMT measures of this cohort are warranted. Lastly, we did
not collect any physical activity data.
In conclusion, our results indicate a high variability in the magnitude of CIMT
change in overweight Latino youth. Fasting insulin measures and glucose effectiveness
appear to contribute to CIMT progression but LDL-cholesterol was the sole predictor of
CIMT progression. These finding extend our previous findings where persistent
metabolic syndrome, high blood pressure and high waist circumference were related to
CIMT and suggest that different cardiometabolic risk factors contribute to CIMT or its
progression in overweight Latino youth.
80

Table 5-1: Descriptive table of baseline physical and metabolic characteristics by
gender

Males Females p-value
Baseline Characteristics (n= 38) (n= 34)
Age (years) 14.5 ± 1.6 14.6 ± 1.8 NS
Maturation stage (by Tanner) NS
1/2 8 0 0.09
3 6 8
4/5 24 26
Body composition
Height (cm) 166.8 ± 9.5 158.6 ± 5.8 <0.001
Weight (kg) 87.1 ± 24.0 81.4 ± 20.4 NS
BMI (kg/m
2
) 31.1 ± 7.2 32.1 ± 6.5 NS
BMI z-score
Total Lean Tissue Mass (kg) 54.0 ± 11.2 45.8 ± 8.6 0.001
Total Fat Mass (kg) 26.5 ± 11.2 31.4 ± 11.6 0.08

Cardiometabolic risk factors
Presence of metabolic syndrome 11 (29%) 5 (15%) NS
Waist Circumference (cm) 93.9 ± 14.5 91.5 ± 13.1 NS
Systolic blood pressure (mmHg) 119.0 ± 10.1 110.8 ± 8.9 0.001
Diastolic blood pressure (mmHg) 65.7 ± 5.3 62.5 ± 5.6 0.02
HDL Cholesterol (mg/dL) 35.3 ± 7.3 39.9 ± 9.2 0.02
Triglycerides (mg/dL)
108.7 ± 54.2 99.1 ± 44.8 NS
LDL-cholesterol (mg/dL) 89.0 ± 21.8 80.7 ± 20.7 NS
Total cholesterol (mg/dL) 146.0 ± 24.5 140.4 ± 24.4 NS

Fasting Glucose (mg/dL) 89.7 ± 7.3 86.7 ± 7.7 0.09
2-hr Glucose (mg/dL) 113.4 ± 19.5 120.7 ± 22.7 NS
HbA1c (%) 5.4 ± 0.3 5.2 ± 0.3 0.07
Fasting Insulin (µU/mL) 14.9 ± 11.5 14.1 ± 7.4 NS
Insulin AUC (nmol/min/L) 305.0 ± 225.4 314.5 ± 194.7 NS
Insulin sensitivity ((x10
-4
/min
-
1
)/µU/mL))
1.78 ± 1.2 1.52 ± 0.78 NS
Acute Insulin Response (µU/mL)
-1
1798 ± 1277 1298 ± 778 0.06
Disposition index (x10
-4
/min
-1
) 2250 ± 940 1705 ± 906 0.02
Glucose effectiveness (% per min) 0.015 ± 0.007 0.017 ± 0.008 NS

Sub-clinical measures of
atherosclerosis

CIMT (mm) 0.598 ± 0.071 0.612 ± 0.043 NS
Maximum diameter (mm) 7.45 ± 0.68 7.18 ± 0.38 0.046
Minimum diameter (mm) 6.46 ± 0.62 6.27 ± 0.36 NS

81

Figure 5-1: Baseline glucose effectiveness is negatively correlated to CIMT
change

Legend: Partial correlation is adjusted for gender, baseline CIMT, age, and height.
82

Figure 5-2: Participants with advanced CIMT progression had significantly higher
baseline LDL- and total cholesterol

Legend: ANCOVA is adjusted for gender, baseline age, Tanner stage, CIMT, HDL-
cholesterol and height.
P<0.05
*
*
83
Table 5-2: Determinants of CIMT progression and advanced progression using
multivariate logistic regression

OR 95% CI P-value

Model 1: Predictors of CIMT Progression
Male 0.83 0.23-3.09 0.79
Age 0.98 0.66-1.44 0.91
Height 0.92 0.84-1.01 0.07
Baseline CIMT (per 0.1mm) 0.50 0.00-1.5 0.14
Fasting Insulin 1.02 0.96-1.08 0.63
Glucose Effectiveness (per 0.001%/ min) 0.50 0.00-2.5 0.57
LDL-cholesterol 1.03 1.003-1.06 0.03

Model 2: Predictors of Advanced CIMT
Progression

Gender 2.90 0.72-11.74 0.14
Age 1.11 0.75-1.65 0.59
Height 1.01 0.93-1.17 0.69
Baseline CIMT (per 0.1mm) 0.02 0.00-5.8 0.24
Fasting Insulin 1.00 0.94-1.07 0.99
Glucose Effectiveness (per 0.001%/ min) 2.86 0.00-2.0 0.79
LDL-cholesterol 1.03 0.99-1.06 0.06

84
CHAPTER 6: SUMMARY OF FINDINGS, FUTURE DIRECTION AND CONCLUSIONS
This chapter begins with a summary of findings, followed by an analysis of
potential underlying mechanisms. The next section will address several strengths and
limitations of our research question and study design. Based on the aforementioned
summary, a section of future studies will be addressed. The chapter closes with the
overall conclusion of this dissertation.
Summary of findings
The purpose of this dissertation was to assess sub-clinical atherosclerosis risk in
overweight Latino adolescents. The overarching hypothesis was that in these children
at high risk for type 2 diabetes, the metabolic syndrome and related mechanisms such
as abdominal adiposity and insulin resistance would be associated with CIMT. Prior to
designing the primary research questions, a preliminary analysis was performed to
determine possible cardiometabolic correlates of CIMT. In a cross-sectional analysis,
impaired fasting glucose was an important correlate of CIMT and systolic blood
pressure was the sole independent predictor of CIMT. These findings suggest that
impaired fasting glucose and systolic blood pressure are important components in
atherosclerosis risk assessment of overweight Latino adolescent youth. The main
objectives of this dissertation were then addressed in three papers. In the first paper,
the objective was to determine the effects of persistent metabolic syndrome over a 3-
year period on CIMT. For the second paper, the objective was to examine the temporal
patterns of abdominal adiposity and insulin resistance that influenced CIMT. The
objective of the third paper was to examine the progression of CIMT and its
cardiometabolc predictors that contributed to CIMT change over a 2-year period.
In paper 1, we showed that children with persistent metabolic syndrome over a
3-year period had a 7% higher CIMT compared to those who never had metabolic
85
syndrome. This finding remained significant after adjusting for covariates including
gender, baseline age, total body fat mass, total lean tissue mass and insulin sensitivity.
An analysis of the individual metabolic syndrome components revealed that persistent
high waist circumference and persistent high blood pressure were associated with
increased CIMT, although these effects may be explained by baseline total body fat and
lean tissue mass.
In paper 2, we hypothesized that increasing visceral adiposity and decreasing
insulin resistance over a 3-year period would be associated with higher CIMT. A
significant interaction between the CIMT tertile groups was observed with visceral
adiposity although it was not easily interpretable. Insulin sensitivity declined in all
participants over the 3-year period; there was no clear relationship between any
measure of insulin resistance and CIMT. Children in the highest CIMT tertile
maintained constant levels of fasting glucose whereas the children in the lower tertiles
of CIMT had a 3-4% decrease in fasting glucose.
In paper 3, the results indicate that change in CIMT was highly variable; 53% of
the participants exhibited an increase in CIMT while the remaining 47% of participants
showed a decrease in CIMT. Moreover, 36% of participants showed progression
beyond that of the physiological norm (Advanced CIMT Progression Group with CIMT
!0.01mm over 2 years). Participants in the advanced CIMT progression group had
significantly higher baseline LDL and total cholesterol than those in the CIMT non-
progression group. Another predictor of CIMT change was baseline glucose
effectiveness, which was negatively correlated with CIMT change independent of
gender and baseline CIMT, age and height. Multivariate logistic regression showed that
the odds of CIMT progression increased 1.03 times (or 3%) for each 1mg/dL increase of
86
baseline LDL-cholesterol, independent of glucose effectiveness and other covariates.
These results highlight the potential effects of LDL-cholesterol in youth on advanced
CIMT progression.
Table 6.1 is a summary of results. It is important to note that cardiometabolic
predictors of baseline CIMT and CIMT change did not clearly overlap. This suggests
that different risk factors may work in concert to cause damage and thickening to the
carotid artery of overweight Latino children. We hypothesize several potential
mechanisms for these observations.

Table 6-1: Summary of results from all studies
Predictor of baseline CIMT? Predictor of CIMT change or
progression?
Variables related to insulin
resistance

Insulin sensitivity No; decline of SI observed in
all groups
No
Visceral adiposity Some evidence of higher
visceral adiposity in those with
higher CIMT
No
Metabolic syndrome Yes; persistent MetS over 3-
years predicted CIMT
No
Variables associated with
hyperglycemia

Fasting glucose Yes; higher levels of fasting
glucose associated higher
CIMT
No
Glucose effectiveness No Yes; negative relationship
between glucose
effectiveness and CIMT
change
Components of the
metabolic syndrome

Blood pressure Yes; persistent high BP
predicted CIMT
No
Waist circumference Yes; persistent high waist
predicted CIMT

Other variable associated
with CIMT

LDL-cholesterol No Yes; sole predictor of CIMT
progression

87
Mechanisms underlying the inter-relationships of hyperglycemia, glucose effectiveness,
and CIMT
There were no relationships between insulin sensitivity and CIMT or CIMT
progression. Instead, we reported that children in the highest CIMT tertile had a 3%
higher fasting glucose in the preceding 3-year period, when compared to those with
lower CIMT. In addition, there was a negative correlation between baseline glucose
effectiveness and CIMT change over a 2-year period, independent of covariates.
Together, these findings highlight the importance of hyperglycemia and glucose-
mediated systems and their effects on CIMT and atherosclerosis development.
First it is important to be familiar with the factors that compose glucose
tolerance: the circulating level of insulin, the insulin sensitivity of tissues and the ability
of glucose to promote its own disposal. Glucose homeostasis depends on the proper
regulation and interaction of these factors and hyperglycemia occurs when there is a
defect in any of these three factors.
26, 50
When insulin is at basal levels, the effect of
glucose is the primary determinant of its own disposal. Specifically, glucose
effectiveness is defined as the ability of glucose to both suppress endogenous glucose
production and stimulate glucose uptake.
31
Glucose effectiveness can be responsible
for as much as 80-99% of glucose tolerance in patients where insulin-mediated systems
are failing (i.e., insulin resistance) or have failed (i.e., type 2 diabetes).
26
When glucose
effectiveness is dysfunctional, this can lead to worsening hyperglycemia, as shown
repeatedly in type 2 diabetics.
72

The relationship of hyperglycemia and glucose effectiveness has not been
extensively studies in non-diabetics. In this cohort, there was a negative relationship
between glucose effectiveness with fasting glucose (r= -0.31, p=0.01) and hemoglobin
88
A1C (r= -0.27, p=0.04), a marker of hyperglycemic levels from the previous 3 months. It
is not surprising to see a relationship between glucose effectiveness and hyperglycemic
measures in a group of overweight and highly insulin resistant adolescents. In fact, this
group of children was an ideal group to study glucose tolerance as some of these
children were already pre-diabetic or had glucose levels on the threshold of pre-
diabetes. This is a critical point because only under these conditions does glucose
effectiveness significantly contribute to glucose disposal.
Yet another interesting point with regard to glucose effectiveness was its
negative correlation to waist circumference (r= -0.26, p=0.03) and visceral adiposity,
(although this relationship was only marginally significant r= -0.19, p=0.09). The link
between abdominal adiposity and glucose effectiveness could be due to lipotoxicity (an
increase in free fatty acids released from visceral adipose tissue). The literature has
shown that increased hepatic insulin resistance and a decline or loss of hepatic glucose
effectiveness is due to an increase of free fatty acids.
73, 92
Elevated fatty acids have
been shown to impair glucose effectiveness via rapid metabolic fluxes of hepatic
enzymes, glucokinase and glucose-6-phosphatase.
92, 98
These are key enzymes in
hepatic carbohydrate metabolism.
112
Alteration of this pathway in type 2 diabetic
patients, for example, shows that a decrease in glucokinase and an increase in
glucose-6-phosphatse are associated with increased fasting glucose production and
failure of glucose to suppress hyperglycemia.
41
This explains the role of free fatty acid
release by visceral adipose tissue. Since visceral adipose tissue releases more free
fatty acids compared to other types of adipose tissue
173
, this may explain the negative
correlation between abdominal adiposity and glucose effectiveness. Originally, we
hypothesized that visceral adiposity would be associated to CIMT via increased hepatic
89
insulin resistance but instead it appears that visceral adiposity may contribute to
glucose effectiveness.
Now we can discuss the implications of altered glucose effectiveness on CIMT
progression. We can speculate that a disruption of glucose homeostasis may trigger a
physiological mechanism that contributes to arterial wall dysfunction. Hyperglycemia
causes oxidative stress that initiates a chain of events, with the inception of superoxide
anion (a prominent ROS) overproduction. Hyperglycemia is thought to initiate this chain
by increasing superoxide anion production via the electron transport chain (with NADH
dehydrogenase at complex I and at the interface of ubiquione and complex III) in the
inner membrane of the mitochondria.
119
This overproduction of superoxide anion is
implicated in increased intracellular production of AGEs, which are modifications of
lipids (or proteins) caused by non-enzymatic oxidation or glycation after contact with
aldose sugars.
63
The accumulation of AGEs can affect vascular cell function by
modifying extracellular and intracellular function with the interaction of AGEs with AGE
receptors.
34
It has also been shown that binding of glycated LDL-cholesterol to LDL-
cholesterol receptors on the endothelial cell surface reduces nitric oxide production and
can suppress uptake and clearance of LDL-cholesterol.
60
AGEs can also interact with
endothelial RAGEs that initiates signals in the MAP-kinase pathways or the GTPase
pathways (CDC42 and Rac) that up-regulate the transcription factor, NF-%B. This can
then transcribe target genes including those for IL-6, TNF-", and VCAM-1, among
others.
16
All of these factors may contribute to inflammation and thickening of the
arteries.
Although we cannot determine the relationships between AGEs and associated
inflammatory factors with CIMT with the current data, we were able to show that
90
children with advanced CIMT progression had increased levels of LDL-cholesterol. We
could further confirm if this hypothesized mechanism holds true by testing the remaining
blood samples of this cohort for glycated LDL-cholesterol and inflammatory markers
associated with AGE activation. This will be further discussed in the future studies
section.
Mechanisms underlying the inter-relationships of high blood pressure, LDL-cholesterol,
and CIMT
Persistently high blood pressure predicted a higher CIMT and LDL-cholesterol
was a predictor of CIMT progression. High blood pressure is thought to cause
hemodynamic damage to the arterial walls while LDL-cholesterol can accumulate as a
response to injuries on the walls. We hypothesize that high blood pressure over time in
these children may have caused a disruption in the endothelium’s ability to respond to
shear stress.
Our data suggest that the relationship of persistently high blood pressure
associated with higher CIMT may be a result of an inability to properly respond to shear
stress incurred on the endothelial wall. Studies have shown that atherosclerotic lesions
occur at sites of greatest hemodynamic insult (areas of low shear stress), with
preference towards the inner wall curvatures and outer wall of bifurcations.
140
It has
been proposed that these sites have higher vulnerability to AGEs (or modified LDL-
cholesterol) due to their exposure to a lower wall shear rate and then incur physical
arterial wall damages.
14
In this study we measured CIMT at 1 cm below the carotid
bifurcation, a highly vulnerable area with low shear stress. Determining if the area of
CIMT measurement is one of high or low shear stress is beyond the scope of this
dissertation. However, if deterioration of the endothelial wall was caused by higher
91
blood pressure, we could theorize that lipids, in conjunction with oxygen radicals and
inflammatoy proteins caused increased CIMT. Examples of inflammatory factors that
have been associated with endothelial dysfunction (via decreased nitric oxide in
cultured human cells) are: IL-6, IL-1, TNF-", and NF-%B.
177
These same inflammatory
factors have also been implicated in hypertension.
17

The relationships between various lipids and CIMT have been examined in
cross-sectional studies of children, yet no clear associations have been established.
12,
13, 18, 137, 175
Overweight and insulin resistant children have been shown to have
decreased HDL-cholesterol and elevated triglyercides, but they do not always exhibit
high levels of LDL-cholesterol.
154
Based on this literature, it was of interest to find that
even slight increases in LDL-cholesterol were associated with CIMT progression.
Further study of dyslipidemias is vital. Cholesterol levels measured longitudinally
are prominent risk indicators of future cardiovascular disease events.
78, 100
There have
been three large-scale longitudinal studies focusing on the relationship between CVD
risk factors in childhood and their long-term effects (1-3 decades later) on CIMT: The
Bogalusa Heart Study, The Muscatine Study and The Young Finns Study. These
investigators found that in addition to obesity and blood pressure, LDL-cholesterol was
one of the best predictors of elevated CIMT and cardiovascular disease events into the
fourth decade of life.
21, 49, 87, 100, 133

Strengths and associated clinical relevance
The strengths of this study include its longitudinal measures of subclinical
atherosclerosis using the same sonographer and reader of the ultrasound images for all
baseline and follow-up CIMT measures. Assessment of cardiovascular risk was done
with clinical measures of total and regional body composition (DEXA and MRI scans)
92
and direct measures of insulin sensitivity (FSIVGTT with minimal modeling). The use of
a large homogeneous sample of understudied minority youth contributes to the
strengths of this report.
The use of clinically applicable measures of fasting blood measures with current
clinical cut-offs for diagnoses of pre-diabetes, high blood pressure, and dyslipidemias
made the results of this dissertation clinically relevant. The clinical significance of this
dissertation is two-fold: First, cardiometabolic correlates of CIMT will serve as important
clinical markers for subclinical atherosclerosis in overweight Latino children. Second,
CIMT could be a useful tool for clinicians, as it may with the identification of children at
high-risk for cardiovascular diseases. Our results support the American Heart
Association recommendation for the use of CIMT in conjunction with traditional risk
factor assessment (such as risk factors we reported: blood pressure, waist
circumference and fasting glucose). Children identified as high-risk could potentially
benefit from early and aggressive behavioral interventions. Moreover, the results could
be extended to either support or reassess pharmacological interventions, such as those
recommended by the American Academy of Pediatrics, which now supports the use of
statins for overweight youth at risk for dyslipidemias. Although our results do show that
a single measure of LDL-cholesterol can predict CIMT progression, the levels of LDL-
cholesterol in these children were not abnormally high. Our results certainly bring
attention to the importance of cholesterol surveillance in overweight children but do not
necessarily support the use of pharmacological interventions.
Limitations of the study design
Several limitations should be addressed. The first limitation is the issue
of a relatively small sample and the implications on statistical power. The data for this
dissertation was collected from a research project designed to assess risk for type 2
93
diabetes. A total of 123 subjects were recruited in 2006 for CIMT collection that were
part of the original cohort (recruited in 2001-2003). Due to this restricted pool of
participants, an a priori sample size analysis was not completed. Instead, we calculated
the minimum detectable power for the sample size used in each analysis (see Chapter
1). In cases where we did not see a significant relationship, for example with adiposity
measures and CIMT, a post-hoc analysis revealed that our sample sizes were
underpowered (<80%).
The second limitation was related to the distribution of the outcome variables,
CIMT and BMI. In our cross-sectional results, we observed that 98% of participants had
a CIMT 10% to 200% higher than the normative values in Caucasian children
88
, with
most of CIMT measures clustered at the 0.600mm measurement. The elevated mean
CIMT of the cohort could be attributed to many factors such as the high BMI, ethnicity or
the family history of type 2 diabetes. This brings us to the distribution of BMI, which
clustered around the 95
th
percentile of BMI. This was an important indicator that a lean
control group would aid in discerning any important differences in adiposity and how
they relate to CIMT. Without separate groups of lean controls, other ethnicities and/or
participants without family history of type 2 diabetes, we accepted that it would be
difficult to uncover any relationships with CIMT in this cohort.
Our third limitation arose when we found that persistent high blood pressure and
the progression of hypertension were associated with CIMT. In our current design we
do not have complete family histories of all participants for hypertension or
cardiovascular disease history, hence it is a limitation. Epidemiological studies have
shown that adiposity and ethnic-linked genetics are highly correlated with elevated
blood pressure. Population studies have reported that since 1980, there has been a
94
parallel between increasing BMI and increasing blood pressure.
116, 124
As for genetics,
the results of the VIVA LA FAMILIA study of Latino families showed that various
pleiotropies may be linked to obesity and its related co-morbidities, including systolic
blood pressure with fasting glucose.
36
In addition, recent evidence of common genetic
determinants between sub-clinical atherosclerosis and high blood pressure have also
been reported in hypertensive Latino families.
40, 176
Hypertension or a family history of
hypertension could then be considered a contributor of elevated CIMT in our study and
adjusting for this covariate either in the statistical analyses is warranted.
The fourth limitation of this study is with regard to limited data available on
inflammatory factors and adipokines, therefore our inability to properly address their role
in the hypothesized relationship between insulin resistance and CIMT. Inflammatory
responses also occur with endothelial dysfunction. Cytokines, namely interleukin-6 (IL-
6), have been shown to increase in obesity and insulin resistance and are closely linked
to endothelial dysfunction and inflammation. IL-6 can either act directly on the
endothelium or by increasing IL-1 and tumor necrosis factor-alpha (TNF-"), which then
increase the expression of NF-%B. The acute phase reaction is an inflammatory process
that initiates secretion of cell mediators at the site of lesions and then mobilizes a
systemic response. In atherosclerosis, these inflammatory acute phase reactions
increase the circulation of C-reactive protein and fibrinogen.
42, 127
C-reactive protein is
the main acute phase protein and has been shown to bind to cell membrane
components (following rupture) to eventually cause break-down and removal of these
structures from the circulation.
115
This is important, as many of the adipokines secreted
by adipose tissue have been associated with dysfunction of the endothelium and
eventually lead to atherosclerosis. The adipokines that are secreted by adipose tissue
95
and are implicated in progression of atherosclerosis are adiponectin
153, 161
, leptin
155
and
cytokines such as IL-6
9
and TNF-".
62
Although we have this adipokine data on some
visits in SOLAR, we have no data for the 2006 calendar year.
Limitations of the Metabolic Syndrome Definition
There are two main weaknesses in our current analyses of the metabolic
syndrome with CIMT. First, there is no consensus on the metabolic syndrome
definitions and it may be that our definition is not the most suitable for testing CIMT as
an outcome. Second, defining excess cardiometabolic risk of the metabolic syndrome
components by using values above specific thresholds could exclude subjects at
moderate risk. In this case, progressive tracking of each component gives a more
comprehensive view of changing cardiovascular risk profiling.
In the cross-sectional analysis (in Chapter 2 of the dissertation), we showed no
significant differences in CIMT by metabolic syndrome status. Only two studies have
shown this relationship in cross-sectional studies of children.
81, 137
Reinehr et al.,
137

compared four different definitions of the metabolic syndrome in children. Only two
metabolic syndrome definitions (Viner et al. and Weiss et al.,
169, 171
showed significant
relationships with CIMT. Based on this detailed report, we can deduce that the different
metabolic syndrome definitions may affect our results. In fact, this has been a highly
debated topic and there has been no consensus on assigning the appropriate cut-offs
for each metabolic syndrome component.
152
The most notable differences between the
four pediatric definitions are the criteria for dyslipidemias, hyperglycemia and abdominal
adiposity (see Table 6-1). The definitions of Cruz et al.
46
and Cook et al.
43
use age and
gender-adjusted cut-off of dyslipidemias, while Viner et al. and Weiss et al. use
universal cut-offs. With regard to hyperglycemia categorization, Cruz et al. and Weiss
et al. both use impaired glucose tolerance, while Cook et al, uses impaired fasting
96
glucose and Viner et al. uses either impaired glucose tolerance, impaired fasting
glucose or high fasting insulin levels by pubertal status. Finally, for high adiposity levels,
waist circumferences >90
th
percentile cut-offs by age, gender and ethnicity are used by
Cruz et al., while Cook et al. used their own dataset for the >90
th
percentile cut-off.
Both Viner et al. and Weiss et al., used >97
th
percentile BMI rather than waist
circumference. These differences in categorizations of dyslipidemias, hyperglycemias
and high BMI/waist circumference may have an impact on any study hypothesizing
relationships with the metabolic syndrome.
Table 6-2: Criteria for 4 pediatric definitions of the metabolic syndrome (adapted
from Shaibi & Goran, 2008 and Reinher et al., 2008)
MetS Def 1
46
MetS Def 2
43
MetS Def 3
169
MetS Def 4
171

Cruz et al.
!3 of 5 criteria
Cook et al.
!3 of 5 criteria
Viner et al.
!3 of 4 criteria
Weiss et al.
!3 of 5 criteria
Adiposity !90
th
percentile
for Waist
circumference
(by ethnicity)
!90
th
percentile
for Waist
circumference
>97
th
percentile
for BMI
>97
th
percentile
for BMI
Elevated Blood
Pressure
!90
th
percentile
for age, gender,
height
!90
th
percentile
for age, gender,
height
!90
th
percentile
for age, gender,
height
!95
th
percentile
for age, gender,
height
Hyperglycemia IGT IFG IFG or IGT or
impaired fasting
insulin
IGT
HDL-cholesterol $10
th
percentile
for age, gender
$40 mg/dL $35 mg/dL $5
th
percentile
for age, gender,
ethnicity
Triglycerides !90
th
percentile
for age, gender
!110 mg/dL !150 mg/dL
OR
!200 mg/dL
total cholesterol
!95
th
percentile
for age, gender,
ethnicity

Following the design by Reinher et al., we recoded the metabolic syndrome
according to three additional definitions of the metabolic syndrome for children (total of
97
four definitions). Using an unadjusted one-way ANOVA (Figure 6-1), there were no
significant differences in CIMT by number of metabolic syndrome components with any
of the proposed definitions. There was however, a trend (p<0.1) for the increasing
number of metabolic syndrome components associated with elevated CIMT by the
Viner et al. definition, that uses either impaired fasting glucose or impaired glucose
tolerance for hyperglycemia and also uses the >97
th
percentile for BMI rather than the
waist circumference cut-off. This less stringent definition may be a better predictor for
CIMT in our cohort. We could extend these analyses to the persistence of the
metabolic syndrome and CIMT for further exploration.

Figure 6-1: Mean CIMT using 4 different metabolic syndrome definitions, stratified
by number of components

0.52
0.54
0.56
0.58
0.6
0.62
0.64
0 1 2 3
CIMT (mm)
Number of MetS components
CIMT by MetS Definitions
MetS
Cruz
MetS
Cook
MetS
Viner
MetS
Weiss
98

High systolic blood pressure was associated with CIMT in the cross-sectional
analyses. In our longitudinal analyses, subjects with persistent high blood pressure had
significantly higher CIMT than those with intermittent or those who never had high blood
pressure. This led us to believe that high blood pressure may be driving the results we
reported with persistent metabolic syndrome and elevated CIMT. If this was the case,
we must consider the possibility that 0.047mm increase in CIMT is caused by medial
hypertrophy caused by high blood pressure (rather than inflammation causes by the
inception of the atherosclerosis process). Furthermore, changes (or thickness of the
intima) have been suggested to be an adaptive response to shear and tensile stress
often caused by hypertension.
32, 52, 106
Of note, we showed that of the 16 participants
who had persistent metabolic syndrome, 6 of them (38%) also had persistent high blood
pressure. We do not know if these children have a family history of hypertension, but
given the high prevalence of hypertension in Latinos, it would be ideal to either control
for family history or design a new study where we can match for family history.
Future Study
The goal of a new study would be to evaluate two proposed mechanisms that
stemmed from the results of this dissertation. First, we established that glucose
effectiveness and hyperglycemia had to work in concert leading to increased CIMT or
CIMT change. We theorized that a hyperglycemic environment might have contributed
to the production of AGEs, causing oxidative stress and damage to the endothelium
ultimately increased CIMT. Secondly, we reported a connection between persistently
high blood pressure and increased CIMT. We theorized that high blood pressure over
time in these children may have caused a disruption in the endothelium’s ability to
respond to shear stress. As a response to injury, this may have triggered inflammatory
99
responses that promote atherogenic mechanisms (such as LDL-cholesterol
phagocytosis) and then arterial wall thickening.
Purpose of the Study and Specific Aims: The goal of this project is to study the
influence of hyperglycemia, high blood pressure, and its mediators on subclinical
atherosclerosis risk in Latino adolescents. Dysfunction of glucose metabolism and
hypertension has been associated with atherosclerosis and vascular disease in adults
however in children these relationships are understudied. Hyperglycemic states and
dysfunction of glucose disposal foster atherogeneic mechanisms via oxidative stress
and formation of AGEs. Hypertension causes mechanical stresses on the arterial wall
that promote inflammatory responses by cytokines such as IL-1, IL-6, and TNF-", which
are also implicated in endothelial dysfunction and increased CIMT. These mechanisms
have not been fully elucidated in either adults or children. Studies in children are sparse
but fasting glucose measures and high blood pressure have been associated with
CIMT. Our laboratory has established that impaired fasting glucose and decreased
glucose effectiveness were associated with increased CIMT or CIMT progression in
overweight Latino children with a history of type 2 diabetes. Independent from these
results, persistent high blood pressure over a 3-year period was also associated with
increased CIMT. Therefore, the overall aim of this cross-sectional study is to examine
the mediating factors between impaired fasting glucose, high blood pressure with CIMT
in overweight Latino adolescents.
Aims and hypotheses:
Aim 1: To assess the role of glucose-mediated mechanisms on CIMT in overweight
Latino adolescents.
100
Hypothesis 1: Overweight pre-diabetic Latino children will have higher CIMT than
normal overweight and normal lean adolescents. This relationship will be mediated by
glucose effectiveness and AGEs (specifically, glycated LDL-cholesterol).
Aim 2: To examine the role of high blood pressure and associated inflammatory
responses on CIMT in overweight Latino adolescents.
Hypothesis 2: Overweight Latino adolescents with high blood pressure will have higher
CIMT than normal overweight and normal lean adolescents. This relationship will be
mediated by inflammatory factors such as IL-1, IL-6 and TNF-".
Significance of new study: This new study goes beyond the scope of the
previous studies by adding 2 new components. First, adding participants and stratifying
them by 2 important physiological states that correlated with CIMT: impaired fasting
glucose status or high blood pressure. Next, adding a lean control group is critical to
assess the differences in participant characteristics and CIMT across all groups. This
proposed study would also expand our knowledge on the relationships between
hyperglycemia, hypertension and atherosclerosis progression in Latino adolescents.
Research Methods and Design

We propose to collect data on 80 Latino adolescents for an observational, cross-
sectional study design. Based on our previous studies, we found significant group
differences in CIMT of 0.047mm in groups with unequal sample sizes. This study would
aim to correct that by recruiting equal numbers of participants with impaired fasting
glucose and high blood pressure. Two control groups of equal sample sizes would also
be added. Since studies of subclinical atherosclerosis in children are limited, identifying
a meaningful change in CIMT has not been standardized. We used G*Power power
program to calculate the sample sizes for independent t-test analyses to detect a
101
0.04mm CIMT difference between group. The calculation was performed with 80%
power and a 5% (two-sided) level of significance. Based on this calculation, we require:
1) a group of 64 overweight adolescents with high blood pressure,
2) a group of 64 overweight adolescents with high fasting glucose,

3) a control group of 64 overweight adolescents without high blood pressure or
high fasting glucose, and
4) a control group of 64 lean, healthy adolescents.
Recruitment and Clinical Criteria

We will recruit 80 Latino adolescents meeting the following criteria: ages 14-18
years and Latino heritage (both parents and grandparents Latino by self-report). Upon
consent, participants will attend a screening visit to assess clinical criteria for placement
into one of the four study groups.
Inclusion criteria:
1) BMI: Participants will have weight and height measurements for BMI
calculation based on 2000 CDC standards. Those with a BMI !85
th

percentile will qualify for 3 of the 4 study groups. Those below the 85
th

percentile may still participate in the lean control group if the capacity for that
group has not been met.
2) Maturation: Participants will have a full medical examination and a licensed
medical staff will assess pubertal maturation. Only participants in post-
puberty (Tanner stage 4 or 5) will be allowed to continue in the study.
3) Blood pressure: After 5 minutes of rest in a sitting position, participants will
have blood pressure readings taken in triplicate and then averaged. A
licensed medical staff will assess if participant has high blood pressure.
102
Participants who are overweight and have high blood pressure will be
assigned to the respective study group. If the participant has a high blood
pressure and has a BMI below the 85
th
percentile, they will be excluded from
the study.
4) Impaired fasting glucose: Participants will have a fasting blood draw to
determine their fasting glucose levels using a glucometer. If the participant
has a fasting blood glucose !100mg/dL and does not have high blood
pressure, they will be placed in the impaired fasting glucose study group.
The result will be verified by the official lab results. If a participant does not
have impaired fasting glucose, they may still qualify for another study group,
dependent on the previous inclusion criteria.
Exclusion criteria:
1) Diabetes: All children will be screened with an oral glucose tolerance test,
and would be free from type 1 and type 2 diabetes. Children who are not
eligible include those with symptoms of polyuria, polydipsia with or without
unexplained weight loss, fasting plasma glucose>126 mg/dl, or a 2-hour
plasma glucose >200 mg/dL during an oral glucose tolerance test. Children
with IGT (2-hour plasma glucose >140 mg/dl during an OGTT) and/or
conditions associated with insulin resistance (e.g. acanthosis nigricans,
hypertension, dyslipidemia, PCOS) will not be excluded. If at any time a child
is diagnosed with type 2 diabetes, a repeat OGTT will be performed and the
family will be referred for treatment.

2) Other exclusion criteria: Due to effects of smoking on inflammatory markers
and its effects on cardiovascular disease, any participants who smoke will

103
also be excluded from the study. All subjects taking any medications known
to influence body composition or insulin action or insulin secretion (e.g.
prednisone, ritalin, growth hormone); b) diagnosed with syndromes or
diseases that may influence insulin action and secretion (eg maturity-onset
diabetes of the young, lipoatrophic diabetes, cystic fibrosis), or body
composition and fat distribution (e.g. Cushing syndrome, Down syndrome);
or, c) previously diagnosed with any major illness since birth (e.g. severe
intrauterine growth retardation, chronic birth asphyxia, cancer).
Overview of Data Collection Paradigm: All testing will be performed at the University of
Southern California Hospital (Clinical Trials Unit) and the USC Atherosclerosis
Research Unit. Table 6-3 below describes the testing sequence all subjects (details of
each measure has been previous described in detail):
Table 6-3: Testing sequence
FULL METABOLIC PROFILING
Physical examination and medical history
Blood pressure (in triplicate)
Height, weight and BMI
Maturation (Tanner staging)
Fasting blood collection
Oral glucose tolerance test (to test diabetes)
Body composition by DEXA
Frequently sampled IVGTT
MEASURES
INCLUDED IN
TESTING (2 visits)

CIMT by ultrasound
TIME REQUIRED
One outpatient CTU visit
One overnight CTU visit
COMPENSATION
$100 gift certificate to children and $50 to
parents
104
Conclusion

This goal of this dissertation was to address the role of insulin resistance and
visceral adiposity in subclinical atherosclerosis of overweight Latino youth. We did not
find any clear relationships with insulin resistance or visceral adiposity and CIMT.
Instead we found that persistent metabolic syndrome, a condition driven by insulin
resistance, was associated with elevated CIMT. Children with persistently high blood
pressure, high waist circumference or high fasting glucose had higher CIMT than those
with normal levels. Change in CIMT over a 2-year period was highly variable and it was
negatively associated with glucose effectiveness. LDL-cholesterol was the sole
predictor of more advanced CIMT progression. Our results suggest that different
cardiometabolic risk profiles contributed to CIMT and its progression in overweight
Latino youth. Upon further review, it seemed plausible that the underlying mechanisms
of these relationships may be working in concert to cause damage to the endothelial
wall of the carotid artery. We theorized that high fasting glucose and its effects on the
endothelium via increased AGEs might have mediated the relationship between glucose
effectiveness and CIMT. This is substantiated by the inverse relationship between
glucose effectiveness and fasting glucose levels. Further study would be necessary with
added data for AGEs. We also theorized that the relationship observed between high
blood pressure and higher CIMT could be the result of both medial hypertrophy and
damage to the intima with increased wall shear stress. Injury to the endothelium caused
by increased blood pressure over time could then allow for LDL-cholesterol to penetrate
and dictate the progression of arterial thickening.
This dissertation has generated important findings related to subclinical
atherosclerosis risk in overweight Latino youth. The results are the first to be reported in
this population of the U.S. and warrant further study. With an increasing Latino
105
population, addressing the health issues of this group is critical, particularly when it
relates to accelerated risk of cardiovascular disease. These studies in conjunction with
future study can fill the knowledge gap in the area of atherosclerosis risk in children.

106

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Asset Metadata

Creator Toledo-Corral, Claudia M. (author)

Core Title Subclinical atherosclerosis in overweight Latino youth: influence of cardiometabolic risk factors

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Preventive Medicine (Health Behavior)

Publication Date 10/07/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag adiposity,carotid intima media thickness,childhood obesity,high blood pressure,impaired fasting glucose,insulin resistance,LDL-cholesterol,OAI-PMH Harvest,subclinical atherosclerosis

Place Name California (states), East Los Angeles (city or populated place), Los Angeles (city or populated place)

Language English

Advisor Goran, Michael I. (committee chair), Hodis, Howard Neil (committee member), Mack, Wendy J. (committee member), Watanabe, Richard M. (committee member), Weigensberg, Marc J. (committee member)

Creator Email ctoledo@usc.edu,toledocorral@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3495

Unique identifier UC1489299

Identifier etd-ToledoCorral-4130 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-408621 (legacy record id),usctheses-m3495 (legacy record id)

Legacy Identifier etd-ToledoCorral-4130.pdf

Dmrecord 408621

Document Type Dissertation

Rights Toledo-Corral, Claudia M.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email uscdl@usc.edu

Abstract (if available)

Abstract Cardiovascular disease is the leading cause of death in the general population. Arterial inflammation and damage manifests as arterial thickening and is the pathological basis for atherosclerosis. The first signs of atherosclerosis are thought to begin in childhood and can be assessed using non-invasive ultrasound measures of the carotid intima media thicknees (CIMT). Children with any of the traditional cardiovascular risk factors, such as obesity or the metabolic syndrome may have early signs of undetectable, or subclinical, atherosclerosis. The purpose of this dissertation was to investigate subclinical atherosclerosis risk in overweight Latino youth with a family history of type 2 diabetes. The specific aims of this dissertation were: 1) to evaluate whether persistence of the metabolic syndrome, a clinically relevant tool, was associated with CIMT