Insulin Resistance, Cognitive Health, and Perfusion of the Adolescent Brain
Phase: NA
Conditions: Insulin Resistance
Sponsor: University of Wisconsin, Madison

Insulin Resistance, Cognitive Health, and Perfusion of the Adolescent Brain 
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Title: Insulin Resistance, Cognitive Health, and Perfusion of the Adolescent Brain 
 
 
PI: William G. Schrage, Ph.D., Department of Kinesiology, University of Wisconsin 
 
 
Funding Sponsor(s): National Institutes of Health (NIH), American Heart Association (AHA) 
 
 
 
 
NCT04089332 
 

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Contents 
I. Abbreviations/Acronyms ..................................................................................................................... 5 
II. Key Personnel ................................................................................................................................... 6 
1. PROJECT SUMMARY ....................................................................................................................... 7 
2. SIGNIFICANCE ................................................................................................................................. 8 
2.1. Synopsis ..................................................................................................................................... 8 
2.2. Detailed Rationale ....................................................................................................................... 8 
2.3. Innovation ................................................................................................................................... 9 
3. SPECIFIC AIMS AND PRELIMINARY EVIDENCE .......................................................................... 10 
3.1. Aim 1 ........................................................................................................................................ 10 
3.1.1. Rationale ............................................................................................................................ 10 
3.1.2. Preliminary Data ................................................................................................................. 10 
3.2. Aim 2 ........................................................................................................................................ 10 
3.2.1. Rationale ............................................................................................................................ 10 
3.2.2. Preliminary Data ................................................................................................................. 11 
3.3. Aim 3 ........................................................................................................................................ 11 
3.3.1. Rationale ............................................................................................................................ 11 
3.4. Study Duration .......................................................................................................................... 11 
4. RESEARCH DESIGN AND METHODS ........................................................................................... 12 
4.1. Subject Population .................................................................................................................... 12 
4.1.1. Inclusion Criteria ................................................................................................................ 12 
4.1.2. Exclusion Criteria ............................................................................................................... 12 
4.1.3. Subject Identification and Recruitment ............................................................................... 13 
4.2. Research Sites .......................................................................................................................... 13 
4.3. Experimental Procedures .......................................................................................................... 14 
4.3.1. Pre-Screening Subjects ...................................................................................................... 14 
4.3.2. Screening Subjects ............................................................................................................ 14 
4.3.2.a. Virtual Screening Procedures ....................................................................................... 14 
4.3.2.b. In-Person Screening Procedures .................................................................................. 15 
4.3.3. Study Visits ........................................................................................................................ 16 
4.3.3.a. Cognitive Testing Visit .................................................................................................. 16 
4.3.3.b. MRI Structural Scanning Visit ....................................................................................... 16 
4.3.3.c. OGTT and MRI Scanning Visit ...................................................................................... 17 
4.3.4. Details of Procedures ......................................................................................................... 18 
4.3.4.a. Informed Consent/Assent ............................................................................................. 18 
4.3.4.b. Fasting, and Caffeine, NSAID and Exercise Restriction ................................................ 18 
4.3.4.c. MRI Screening Document ............................................................................................. 18 

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4.3.4.d. Tanner Stage Questionnaire ......................................................................................... 18 
4.3.4.e. Maximal Exercise Test ................................................................................................. 18 
4.3.4.f. Cognitive Tests .............................................................................................................. 19 
4.3.4.g. Magnetic Resonance Imaging (MRI) ............................................................................ 19 
4.3.4.h. Oral Glucose Tolerance Test (OGTT) ........................................................................... 20 
4.3.4.i. Venipuncture ................................................................................................................. 20 
4.3.4.j. Intravenous Catheter ..................................................................................................... 20 
4.3.4.k. Blood sampling ............................................................................................................. 20 
4.3.4.l. Subject Monitoring ......................................................................................................... 21 
4.3.4.m DEXA Scan .................................................................................................................. 21 
4.3.5 Summary of Study Visits and Procedures ........................................................................... 21 
4.4. Confidentiality Protections ......................................................................................................... 21 
4.5. Remuneration ........................................................................................................................... 22 
5. BANKING AND SHARING OF DATA AND BIOSPECIMENS .......................................................... 23 
5.1. Data .............................................................................................................................................. 23 
5.2. Biospecimens ............................................................................................................................... 23 
5.3. Assurances ................................................................................................................................... 23 
6. RISKS ASSOCIATED WITH PROCEDURES .................................................................................. 24 
6.1 Risk Summary ............................................................................................................................ 24 
6.2 Detailed Risks ............................................................................................................................ 24 
6.2.1. Fasting, and Caffeine, NSAID and Exercise Restriction ..................................................... 24 
6.2.2. Maximal Exercise Test ....................................................................................................... 24 
6.2.3. Intravenous Catheter and Venipuncture ............................................................................. 24 
6.2.4. Blood sampling ................................................................................................................... 25 
6.2.5. OGTT ................................................................................................................................. 25 
6.2.6. MRI .................................................................................................................................... 25 
6.2.7. Subject Monitoring.............................................................................................................. 26 
6.2.8. Cognitive Testing ............................................................................................................... 26 
6.2.9. DEXA Scan ........................................................................................................................ 26 
7. DATA AND SAFETY MONITORING PLAN ...................................................................................... 27 
7.1. Monitoring study safety ............................................................................................................. 27 
7.2. Data Monitoring Committee (DMC) ........................................................................................... 27 
7.3. Adverse Event (AE) Reporting .................................................................................................. 28 
7.3.1. Assessment of Attribution ................................................................................................... 29 
7.4. Serious Adverse Event (SAE) ................................................................................................... 29 
7.5. Identifying, Reviewing, and Reporting Adverse Events and Unanticipated Problems to the IRB 31 

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7.6. Expedited Adverse Event Reporting Requirements ................................................................... 31 
7.7. Protocol Deviations ................................................................................................................... 31 
7.8. Internal Audits ........................................................................................................................... 31 
7.9. Minimizing Research-Associated Risk....................................................................................... 32 
7.9.1. Initial screening .................................................................................................................. 33 
7.9.2. Subject Monitoring.............................................................................................................. 33 
7.9.3. Blood Sampling .................................................................................................................. 33 
7.9.4. Protecting the Confidentiality of Participant Data ................................................................ 34 
7.10. Stopping Guidelines ................................................................................................................ 34 
7.10.1. Study Stopping Guidelines ............................................................................................... 34 
7.10.2 Subject Stopping Guidelines ............................................................................................. 35 
8. STATISTICAL CONSIDERATIONS ................................................................................................. 36 
8.1. MRI Data Processing ................................................................................................................ 36 
8.2. Aim 1 ........................................................................................................................................ 36 
8.3. Aim 2 ........................................................................................................................................ 37 
8.3.1. Aim 2 Data Expression ....................................................................................................... 37 
8.3.2. Aim 2A ............................................................................................................................... 37 
8.3.3. Aim 2B ............................................................................................................................... 37 
8.4. Aim 3 ........................................................................................................................................ 38 
8.5. Statistical Summary .................................................................................................................. 38 
9. DATA AND RECORD KEEPING...................................................................................................... 39 
9.1. Data Management .................................................................................................................... 39 
9.2. Assured Confidentiality ............................................................................................................. 39 
9.3. Data Collection Methods ........................................................................................................... 39 
9.4. Retention of Study Records ...................................................................................................... 40 
10. DATA INTEGRITY ......................................................................................................................... 41 
11. REFERENCES .............................................................................................................................. 42 
12. SUPPLEMENTAL INFORMATION ................................................................................................ 47 
 
 
 
 

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I. Abbreviations/Acronyms 
 
ASL- Arterial Spin Labeling 
AE- Adverse Event 
CBF- Cerebral Blood Flow 
CP- Cerebral Perfusion 
CRF- Case Report Form 
CRU- Clinical Research Unit 
DEXA- Dual Energy X-Ray Absorptiometry 
DMC- Data Monitoring Committee 
ECG- Electrocardiogram 
eCRF- Electronic Case Report Form 
HERC- Human Exercise Research Core 
IR- Insulin Resistance 
IV- Intravenous 
MR- Magnetic Resonance 
MRI- Magnetic Resonance Imaging 
NSAID- Non-Steroidal Anti-Inflammatory Drug 
OGTT- Oral glucose tolerance test 
PC VIPR- Phase-Contrast Vastly-undersampled Isotropic Projection Reconstruction 
REDCap- Research Electronic Data Capture 
RPM- Revolutions Per Minute 
SAE- Serious Adverse Event 
SOP- Standard Operating Procedure 
VCO2- Carbon Dioxide Production 
VO2- Oxygen Consumption 
WIMR- Wisconsin Institute of Medical Research 
 
 
 

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II. Key Personnel 
 
William G. Schrage, Ph.D.- Professor, Department of Kinesiology, University of Wisconsin-Madison 
 
Aaron T. Ward, M.S.- Graduate Student, Department of Kinesiology, University of Wisconsin-Madison 
 
Aaron Carrel, M.D.- Professor, Department of Pediatrics, University of Wisconsin Children's Hospital 
Pediatric Endocrinology, Diabetes, and Fitness 
 
Oliver Wieben, Ph.D.- Associate Professor, Departments of Medical Physics and Radiology, University 
of Wisconsin-Madison 
 
Jana Jones, Ph.D., LP- Neuropsychologist and Associate Professor, Department of Neurology, University 
of Wisconsin School of Medicine and Public Health 
 
Bruce Hermann, Ph.D.- Emeritus Professor, Department of Neurology, University of Wisconsin School 
of Medicine and Public Health 
 
Ron Serlin, Ph.D.- Emeritus Professor, Department of Statistics, University of Wisconsin-Madison 
 
 
 

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1. PROJECT SUMMARY 
 
One in five American adolescents are obese, and many of these patients exhibit some level of insulin 
resistance (IR). IR is associated with cerebrovascular disease, reduced memory, attention, and cognition, 
but how IR contributes to these in the course of adolescent brain development is unclear. The goal of 
this proposal is to investigate the extent and by which IR drives reductions in neurocognitive function and 
cerebral blood flow (CBF) in adolescents at elevated risk for poor brain and cerebrovascular health. We 
are testing the overall hypothesis that neurocognitive decline is linked to the severity of IR, as are 
reductions in brain perfusion, due to dysfunctional insulin signaling. We propose to study acute CBF 
control in healthy adolescents (12-18 years) across a range of IR (diabetes excluded) in collaboration 
with expert childhood neuropsychologists who lead a comprehensive cognitive testing protocol. Next, two 
complementary MRI methods (ASL and phase contrast MRI), along with a physiologic stressor to 
challenge CBF, will be used to test whether the impact of IR on brain macrovascular CBF and 
microvascular perfusion is regionally specific, with more negative effects in temporal and parietal lobes. 
These lobes are some of the initial brain areas to change in neurodegenerative diseases, and are 
involved in attention, memory, learning, and cognition. Finally, this study tests whether the degree of 
cognitive decline is linked to the CBF stress response and to severity of IR, and to what extent brain 
hypoperfusion mediates the IR-driven lower cognitive function. This study will provide the first 
comprehensive look at IR in adolescent brains, by utilizing extensive neuropsychological testing and 
state-of-the-art MR imaging, and drawing on interdisciplinary collaborations between developmental 
neuropsychologists, vascular physiologists, and pediatric endocrinologists. These studies are designed 
to uncover fundamental relationships between IR, neurocognitive function, and CBF in these blossoming 
adolescent brains. Exciting preliminary data support the aims—particularly uncovered by a physiologic 
insulin surge—signifying these findings will serve as a foundation for prospective, mechanistic studies to 
reduce the burden of IR and improve brain and cognitive health in this clinically important population. 
 
 
 

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2. SIGNIFICANCE 
 
2.1. Synopsis 
 
One in five American adolescents is obese. Up to half of those are already exhibiting insulin resistance 
(IR), a hallmark of metabolic syndrome and diabetes linked to serious life-altering health disorders, 
including cardiovascular and cerebrovascular disease. In adults, IR negatively affects brain structure and 
function and is reflected in lower regional brain volumes, perfusion, increased white matter 
hyperintensities and abnormal neuropsychological status, especially affecting memory and attention—all 
changes associated with accelerated cognitive and brain aging and increased risk of dementia. In an 
analogous fashion, a limited set of literature suggests adolescents with IR exhibit similar brain changes 
during maturation. We posit then that the brains of obese adolescents are more susceptible to insults of 
IR during rapid brain development, positioning them on an abnormal cognitive trajectory, and 
predisposing them to issues related to learning, behavioral stress responses, and depression. 
 
While the metabolic consequences of IR are well described in adolescence, the impact of IR on their 
neurocognitive status (intelligence, memory, attention, executive function, processing speed) and 
cerebrovascular function and their interactions remains largely unexplored. This is important since in 
addition to its classic role as a metabolic hormone, insulin acts as a vasodilator and supports neurotrophic 
signaling in healthy humans. Therefore, dysfunctional insulin signaling may hold tremendous influence 
over brain health in adolescents during this vital period of brain development. New insight is required to 
understand where, when, and how IR negatively transforms brain health, including whether a dose-
response exists between IR severity and anomalies in brain and cognition. 
 
The long-term goal of this research program is to determine the influence of IR on brain development in 
adolescents through the relationships between neurocognition and cerebral blood supply. The primary 
goal of the current project is to quantify fundamental neurocognitive and cerebrovascular function in 
relation to the severity of IR. Our central hypothesis is that as IR worsens: a) subtle but meaningful 
neurocognitive declines emerge; b) regional brain perfusion is reduced primarily in areas linked to 
learning and memory despite preserved resting global cerebral blood flow (CBF); c) acute insulin surges 
exacerbate regional hypoperfusion, and d) cognitive scores will be lower, mediated in part by insulin-
stimulated hypoperfusion. 
 
2.2. Detailed Rationale 
 
Approximately one in five U.S. adolescents (12-19 years old) suffer from obesity (15, 32) and as many 
as half of those may suffer from insulin resistance (IR). IR is associated with reduced regional brain 
volumes, connectivity, perfusion, as well as cognitive deficits (46, 49, 52). Adolescents with IR also exhibit 
additional cardiovascular risk factors including hyperglycemia and hypertension, which are known to 
increase the risk and severity of cerebrovascular and neurocognitive diseases in adults (3, 17, 30, 35, 
43). Exactly how IR influences the relationship between brain perfusion and neuronal function in 
adolescence is undetermined. To limit the potential future social, financial and health burdens associated 
with IR, these children and their physicians need new options, from new research directions, to ensure 
healthy brain development as they grow into adults.  
 
Adolescence is marked by a proliferation of synapses with subsequent synaptic elimination (i.e. pruning) 
along with increases in white matter formation and concomitant decreases in grey matter density (1, 6), 
primarily in the prefrontal and parietal cortices. Emerging evidence suggests adolescents with IR exhibit 
reduced brain volumes and connectivity, temporally associated with changes in cognitive abilities, 
particularly related to reductions in executive function (5). Insulin signaling pathways are important to 
neuron growth and development (2, 3, 7, 8, 18, 22, 31), and insulin transport into the brain is dysfunctional 

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in subjects with IR (30). Consistent with insulin’s vital role in brain health, intranasal insulin increases 
brain perfusion (38), and in older adults with IR intranasal insulin improves cognitive outcomes (9). In the 
rapidly proliferating adolescent brain, disrupted insulin delivery or transport foretells of harmful lifelong 
impacts of IR on brain and cognitive development. In adolescents, a key question is whether worsening 
IR contributes to an abnormal neurocognitive trajectory.  
 
Despite the associations of IR with poor brain health, the cerebral blood flow (CBF) response to acute 
insulin surges in humans remains largely unexplored. In healthy blood vessels, insulin evokes 
vasodilation via endothelial nitric oxide synthase (NOS) activation. However, insulin also induces the 
potent vasoconstrictor, endothelin-1, and IR shifts the balance between NOS and endothelin-1, promoting 
vasoconstriction (20, 21, 55). More importantly, NOS becomes “uncoupled”, producing high levels of 
reactive oxygen species (ROS) (20, 21, 55). Excessive ROS damages the neurovascular unit coupling 
blood supply to metabolism, and drives neuronal dysfunction (19, 21). In adolescents with IR, a key 
question is whether cerebrovascular dysfunction exists at all, and its relationship to neurocognitive 
abnormalities at this vital stage of brain development. 
 
In summary, insulin acts as an endocrine hormone, a neurotrophic signal, and a vasodilator, so it is logical 
that dysfunctional insulin signaling plays a pivotal part in linking cerebrovascular and cognitive 
complications in IR (10). These adolescents are particularly vulnerable, as IR occurs during critical stages 
of neurological development, and they present cardiovascular complications earlier in life and for a longer 
duration (29). Detecting neurocognitive and cerebrovascular dysfunction in its earliest stages in 
adolescents will provide the best opportunities to preserve or restore brain function. Addressing the 
questions posed above, by using dynamic testing, will begin to fill a vital knowledge gap, by concentrating 
on essential aspects of when, where, and how adolescent IR cultivates cerebrovascular and 
neurocognitive dysfunction.  
 
2.3. Innovation 
 
Many youth-focused brain studies emphasize a sole aspect of brain structure, cognitive function, or CBF. 
A limitation of intriguing, but associative studies is that they assess effects of IR on resting measures in 
adolescence, obscuring insight into the daily impact of insulin on brain health (26, 37). These resting 
associations lack high resolution in terms of mechanistic insight, and thus add fuel to a classic chicken 
or egg question: Does vascular dysfunction or neural dysfunction dominate the earliest brain changes in 
adolescents with IR? To address several limitations, this proposal combines three unique areas of 
innovation. First, this proposal combines collaborative expertise in pediatric neuropsychology, pediatric 
endocrinology, and vascular physiology to address the complexities and subtleties of the preclinical 
disease process. A second innovation stems from combining two powerful, complementary brain-imaging 
approaches that quantify brain perfusion and identify the regions most impacted by IR. Phase-Contrast 
Vastly-undersampled Isotropic Projection Reconstruction, (PC VIPR) is a high-resolution, 4D flow 
approach used to quantify macroscopic CBF in all major extracranial and intracranial arteries. Arterial 
Spin Labeling (ASL) measures provide insight into microvascular cerebral perfusion allowing 
identification of focused brain regions impacted by IR. No single measure of flow or perfusion can 
comprehensively assess cerebrovascular function in health or IR; the combination of both MRI 
approaches will greatly enhance insight into CBF regulation in adolescence. Third, clinically relevant 
insulin surges acutely stress brain blood flow regulation, highlighting cerebrovascular dysfunction that is 
not apparent at rest. Indeed, we present novel pilot data indicating significant neurocognitive decline 
along with remarkable cerebrovascular dysfunction, both of which are related to the severity of IR. When 
combined, the technical and conceptual innovations in this proposal will deliver novel insight into the 
severity and regional impact of IR on brain health and cognition in adolescents.  
 

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3. SPECIFIC AIMS AND PRELIMINARY EVIDENCE 
 
Pilot studies in Aim 1 were conducted in 7 adolescents (mean age 14.7 years); 3 with low IR (HOMA-IR 
~3.4) and 4 with high IR (HOMA-IR ~4.6). Five of these 7 were studied for Aims 2 and 3 (2 with low IR, 
3 with high IR). 
 
These data were collected from the awarding of a previous UW Graduate School Fall Competition Grant 
under IRB# 2014-0529.  
 
3.1. Aim 1 
 
To test the hypothesis that adolescents with higher IR will demonstrate subtle but clinically meaningful 
neurocognitive dysfunction, primarily in the domains intelligence, memory, attention, executive function, 
and processing speed. 
 
3.1.1. Rationale 
 
Associations between cognition and IR have not been extensively studied and reports are mixed on 
whether IR is associated with cognitive abnormality (24, 42). A handful of studies suggest youth with 
obesity and IR demonstrate deficits in executive control processes (e.g. attention, memory) and lower 
overall intelligence (46, 47). Although IR has been postulated as being a mediator for such cognitive 
deficits (46, 49), the association between systemic IR and cognition has not been well established in 
adolescents (47, 48). 
 
3.1.2. Preliminary Data 
 
Pilot data indicate similar brain and hippocampal volumes between groups (2.93 vs 2.84 mm3). Cognitive 
testing results did not differ significantly between groups. However, episodic memory, executive function, 
and attention, as well as total composite cognition scores were all quantitatively lower in adolescents with 
high IR. Notably, these smoldering decreases in cognition are directly correlated to HOMA-IR. 
Collectively, these data suggest brain structure remains intact at this stage, yet fundamental memory and 
attention measures are declining in relation to worsening insulin resistance. 
 
3.2. Aim 2 
 
To test the hypothesis that adolescents with higher IR: 
a) exhibit intact resting global macrovascular CBF but reduced microvascular perfusion in key brain 
areas involved in cognition, memory, and learning 
b) will exhibit further perfusion reductions in response to an endogenous insulin surge in contrast to 
increased perfusion in healthy adolescents. 
 
3.2.1. Rationale 
 
Patients with IR and cerebrovascular disease make it difficult to tease out the timing and interactions 
between IR, poor perfusion and neurodegenerative disease (12, 14, 21, 55). Using an OGTT as a “stress 
test” for the brain, may evoke acute reductions in perfusion and elevations in ROS, consistent with the 
concept that a disrupted insulin response directly and indirectly promotes neurocognitive decline, as 
OGTT. 
 

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3.2.2. Preliminary Data 
 
First, total CBF is remarkably similar between groups (PC VIPR). However, adolescents with high IR 
exhibit reduced basal microvascular CP in numerous brain regions, including those involved in cognitive 
processing (ASL). The combination of MRI approaches suggests an uncoupling of macro and 
microvascular flow (17) (34), consistent with early but subtle basal microvascular dysfunction in IR. 
Therefore, it is crucial to look at both CBF and CP measures, as regional regulation appears to be 
differently affected by IR. 
 
Second, following OGTT, regional CBF and CP patterns change markedly. Despite similar increases in 
plasma glucose, insulin increases much more in adolescents with high IR. CBF through the Middle 
Cerebral Artery (MCA) increases in adolescents with low IR but decreases in adolescents with high IR. 
Similar patterns were observed in the Anterior Cerebral Artery (ACA, not shown). Third, 45 minutes into 
OGTT, select regions increased CP in adolescents with low IR, but decreased CP in adolescents with 
high IR. Notably, many of these regions are downstream of the MCA and ACA and are linked with 
executive function, memory, and learning abilities. These data strongly support the hypothesis that 
adolescents with high IR demonstrate overt brain endothelial dysfunction, which decreases brain 
perfusion under the physiologic stress of hyperinsulinemia and hyperglycemia. 
 
In summary, the combination of PC VIPR and ASL MRI approaches allow us to quantify dynamic CBF/CP 
responses, identify brain regions most sensitive to insulin vasodilation, and most impacted by IR. 
Preliminary data for Aim 2 OGTT evokes hypoperfusion occurring primarily in anterior brain regions 
associated with executive function and memory. In the context of insulin-driven ROS production (20, 21), 
and lower cognition scores (Aim 1), these data are consistent with the hypothesis that acute brain 
hypoperfusion, via daily insulin surges, plays an essential role in the early pathology of cognitive 
dysfunction in adolescents with high IR. 
 
3.3. Aim 3 
 
To test the hypothesis that higher IR is associated with lower cognitive function (Aim 1) in such a way 
that perfusion at rest (Aim 2A), and perfusion changes during insulin surge in particular (Aim 2B), mediate 
the relationship. 
 
3.3.1. Rationale 
 
Lower CBF is linked to cognitive decline. OGTT actively reduces CBF/CP in adolescents with high IR, 
activates ROS (20, 21). These multiple daily insults may independently or collectively contribute to poor 
cognitive outcomes during this vital stage of brain development. 
 
3.4. Study Duration 
 
Expected start date: July 1, 2019 
Expected end date: June 30, 2021 
 
 
 
 

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4. RESEARCH DESIGN AND METHODS  
 
The procedures described herein are largely the same or similar as that of past procedures outlined in 
IRB 2014-0529. General procedures that are the same include: 
• 
Pediatric population (healthy and with IR) 
• 
Venipuncture, IV placement, and blood draws 
• 
Cognitive testing 
• 
MRI scans 
• 
OGTT administration 
Procedures that differ include: 
• 
Some changes in inclusion and exclusion criteria 
• 
Addition of DEXA scan 
• 
Addition of maximal exercise test 
• 
More extensive cognitive tests 
4.1. Subject Population 
 
Fifty-five (55) otherwise healthy adolescents, post-puberty, between 12-18 years of age inclusive. 
 
4.1.1. Inclusion Criteria 
 
Subjects will be included if they meet all of the following criteria: 
• 
Age 12-18 years inclusive 
• 
Typically developing and cognitively intact 
 
We do not plan to recruit subjects at specific levels of IR. By the variable clinical nature of IR, subjects 
may display similar glucose levels yet a broad range of insulin values. Subjects will have IR quantified 
directly via the OGTT. The range of IR increases clinical external validity and will provide important pilot 
data for future studies. 
 
4.1.2. Exclusion Criteria 
 
Subjects will be excluded if they meet any of the following criteria: 
• 
Diabetes (≥126 mg dL-1 fasting glucose) 
• 
Insulin treatment or sensitizing drugs 
• 
Diagnosis of kidney, pulmonary, or heart disease 
o Note: Subjects with asthma who are taking or prescribed a daily inhaler medication (i.e., 
daily controller medication) will be excluded. Subjects with only as-needed albuterol 
prescriptions will not be excluded unless their asthma limits their ability to exercise or they 
require albuterol prior to any exercise. 
• 
Current smoking* 
• 
Pregnancy 
• 
Neurological or developmental disorders (e.g., intellectual disability, autism) 
• 
Significant head injury or medical conditions (e.g., concussion, encephalopathy, seizure disorder) 
• 
Inability to undergo the MRI procedure  
• 
Weight less than 94.5 lbs (42.9 kg) to adhere to safety guidelines regarding blood sampling and 
OGTT administration 
• 
Tanner Stage <3 

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• 
Any other circumstance deemed by the PI not addressed above 
 
*Current smoking will be defined as the use of tobacco or nicotine products >5 times in the past 30 days. 
 
4.1.3. Subject Identification and Recruitment 
 
Subjects will be recruited primarily from pediatric and pediatric endocrinology clinics via our collaborator, 
Dr. Aaron Carrel, and his staff in UWHC Pediatric Endocrinology. Additionally, subjects will be recruited 
from the greater Madison, WI community. Subjects may also be recruited from other studies in which our 
collaborating physician is an investigator: 
• 
2011-0214 
• 
2013-1647 
• 
2015-1172 
• 
2018-0974 
These study consent forms included an authorization to be contacted for future research. Only subjects 
who signed the authorization will be contacted. 
 
The following methods may also be used for recruitment: 
• 
Mass email to UW-Madison students and staff 
• 
Recruitment flyers 
o Locations may include: 
 
UW-Madison campus and UW Hospital buildings 
 
Local middle schools and high schools 
• 
Middleton/Cross Plains School District 
• 
Verona School District 
• 
Sun Prairie School District 
 
Local businesses 
 
Local physician offices 
• 
Website postings (e.g. Craigslist, UW Student Job Center) 
 
All recruitment material used will be IRB approved prior to posting. 
 
Enrollment will continue until the target sample size (see section 4.1 Subject Population) completes all 
study procedures. 
 
4.2. Research Sites 
 
The following sites will be used for the following purposes:  
• 
UW Medical Sciences Building 
o Maximal exercise testing 
• 
UW Human Exercise Research Core (HERC) in the UW Nursing School* 
o Maximal exercise testing 
o DEXA Scan 
• 
Clinical Research Unit (CRU)* 
o Screening visit blood draw 
• 
Wisconsin Institute of Medical Research (WIMR) 
o MRI 

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o IV placement 
• 
Waisman Center 
o MRI 
o IV Placement 
• 
UW Hospital 
o Cognitive testing 
 
*This flexibility is requested to prevent potential space usage conflicts in the UW Natatorium. Firstly, other 
currently approved research in the PI’s laboratory utilizes the limited space at the UW Natatorium. 
Secondly, in anticipation of the closing of the UW Natatorium circa summer 2020 and subsequent 
laboratory transition to the Medical Sciences Building. Thus, having the ability to conduct the necessary 
research at the UW HERC in the Nursing School or the CRU will allow the proposed research project to 
continue with minimal interference. 
 
4.3. Experimental Procedures 
 
4.3.1. Pre-Screening Subjects 
 
Delegated study team members will screen patients for eligibility for the study based on medical history, 
medication use and evaluate general safety to undergo MRI (e.g. claustrophobia, metallic implants, etc.). 
Consent/permission will be obtained following a description of the study and prior to survey questions 
either: 
• 
verbally for phone screen 
• 
agreeing to the REDCap survey 
The same script/information sheet will be used for both the phone screening and REDCap survey (i.e. 
the REDCap survey will be discussed with the subject verbally and constitutes the “phone screen”). 
 
Medical records of potential subjects will be reviewed by staff under the supervision of Dr. Aaron Carrel 
from Dr. Aaron Carrel’s clinic(s) for fasting glucose and insulin to determine subject's eligibility for the 
study. This review will occur prior to pre-screening procedures. 
 
These steps will be taken to minimize risk of participation. If an individual does not preliminarily meet all 
inclusion criteria or preliminarily meets any of the exclusion criteria, then the subject will be immediately 
eliminated from consideration for the study and their information will be destroyed (e.g. shredder or digital 
deletion). 
 
4.3.2. Screening Subjects 
 
Screening will be split into two (2) parts: 
• 
Part 1 (virtual): Approximately 1 hour 
• 
Part 2 (in-person): Approximately 0.5-1 hour 
4.3.2.a. Virtual Screening Procedures 
 
Procedures include: 
• 
e-Consent: 
o Subjects and subject’s parent(s) (if subject <18 years) will first be contacted regarding 
their preliminary eligibility and attainment of parental permission (if applicable). A REDCap 
link to the electronic version of the Informed Consent form and Informed Assent form (if 

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applicable) will be sent to the subject and subject’s parent(s) (if applicable). Electronic 
verification and signature/initials will be obtained prior to other study activities. 
• 
Questionnaires 
o Following attainment of consent, subjects and subject’s parents (as applicable) will 
complete the following questionnaires in a virtual format via REDCap: 
 
Health history (via questionnaire) 
 
MRI safety (via questionnaire) 
For participants <18 years old, the pre-screening survey answers will be reviewed with the parent of the 
potential subject to ensure accuracy. Any discrepancies will be documented. Verbal verification of 
accuracy by the parent will be documented. 
 
4.3.2.b. In-Person Screening Procedures 
 
A visit reminder will be provided to potential subjects via phone or email at least 24 hours prior reminding 
them of the following (see Sections 4.2. Research Sites and 4.3.4.b. Fasting, and Caffeine, NSAID and 
Exercise Restriction): 
• 
Location of visit (with general campus directions) 
• 
Fasting directions 
• 
Caffeine, NSAIDs, and exercise restriction 
• 
Inform the study team if any albuterol, inhaled steroids, or systemic steroids were taken within 48 
hours 
Subjects will arrive fasted. 
 
The subject will be screened in-person for the following: 
• 
Pregnancy* 
• 
Height and weight 
• 
Hip and waist circumference 
• 
Blood draw 
• 
Body composition via DEXA† 
• 
Maximal exercise test† 
*Female subjects will be asked whether they might be pregnant in a private setting away from their 
parents, and the information will not be shared with their parents. 
 
†For scheduling reasons and the fact that the data obtained from these procedures do not affect eligibility, 
the DEXA scan and the maximal exercise test can be performed on any of the study visits or on 
independent visits. 
 
The results of the screening procedure will be valid for study inclusion for 180 days. If the subject does 
not complete all study visits within 180 days of screening, another screening procedure will take place. 
 
Other studies in the PI’s lab that investigate the same subject population collect similar screening data 
and utilize many of the same procedures. To reduce subject burden, we will use all applicable screening 
data from the other study’s screening procedure and perform only those procedures that were not 
previously performed. The use of previous data will be documented. The results of the screening 
procedure will be valid for study inclusion for 180 days from date of collection. If the subject does not 
complete all study visits within 180 days of screening, another screening procedure will take place. 
 

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4.3.3. Study Visits 
 
Duration: Approximately 6.5-8 hours total. 
 
The study involves 2-3 visits to the WIMR, Waisman Center, or UW Hospital (see section 4.2 Research 
Sites). Because the data collected in each visit is not dependent on the other visit in a temporal manner, 
the study visits can occur in any order (i.e. one visit does not need to occur before another). Additionally, 
the flexibility of conducting study visits in any order allows enhanced scheduling opportunities for subjects 
and staff. 
 
The “Cognitive Testing” and “MRI Structural Scanning” study visits (see section 4.3.3.a Cognitive Testing 
Visit and 4.3.3.b MRI Structural Scanning Visit) may occur together or independently as best suited for 
the subject and research staff. This flexibility allows the potential for the reduction in the number of study 
visits or the reduction in the time spent at a single visit. 
 
A visit reminder will be provided to potential subjects via phone or email at least 24 hours prior reminding 
them of the following (see Sections 4.2. Research Sites and 4.3.4.b. Fasting, and Caffeine, NSAID and 
Exercise Restriction): 
• 
Location of visit (with general campus directions) 
• 
Fasting directions 
• 
Caffeine, NSAIDs, and exercise restriction 
 
Subjects will arrive fasted prior to study visits. Parent(s)/guardian(s) will be encouraged but not required 
to be present during the study visits (exception: cognitive testing visit). 
 
4.3.3.a. Cognitive Testing Visit 
 
Duration: Approximately 2-3 hours. 
 
Subjects will arrive fasted. 
 
Subjects will undergo formal neuropsychological evaluations led by collaborators Drs. Bruce Hermann 
and Jana Jones and administered by trained personnel. Selected tests (see section 4.3.4.e. Cognitive 
Tests) reflect a broad set of core cognitive domains and related tests, which retain the same item pool 
across the age range to be investigated to facilitate examination of factors that influence cognitive 
development in youth with IR (25, 40, 41, 46, 47). This comprehensive approach aims to maximize our 
ability to detect and interpret subtle functional changes. 
 
There is potential for confounders, such as history of academic problems, depression, ADHD, or anxiety. 
These factors will be assessed through a structured interview with the child’s parent(s)/guardian(s) by an 
interviewer blinded to all cognitive and psychiatric information, and will assist in interpreting cognitive 
results. No cognitive information about the parent(s)/guardian(s) will be obtained; they will solely act as 
informants.  
 
4.3.3.b. MRI Structural Scanning Visit 
 
Duration: Approximately 1 hour. 
 
Subjects will arrive fasted. 
 

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We do not expect differences in brain structure (but will compare statistically), but comprehensive imaging 
will examine potential neuroanatomical differences, and control for brain volume differences between 
subjects.  Subjects will lie supine for approximately 1 hour while various MRI neuroanatomical, CBF, and 
CP scans are performed. Scans may include T1-weighted imaging, Diffusion Tensor Imaging, T2, 
T2FLAIR, MPnRAGE, ASL, and PC VIPR. These scans are well-established methods used by the PI’s 
collaborators in the Wisconsin Alzheimer’s Disease Research Center (4, 17, 43, 44). 
 
4.3.3.c. OGTT and MRI Scanning Visit 
 
Duration: Approximately 2 hours. 
 
Subjects will arrive fasted. 
 
Subjects will have one (1) IV catheter placed (see section 4.3.4.i. Intravenous Catheter for details) and 
then lie supine in the MRI machine. Subjects undergo baseline MRI scans lasting approximately 20 
minutes, following which subjects will be removed from the MRI bore to sit to drink 75g glucose (OGTT; 
see section 4.3.4.g. Oral Glucose Tolerance Test (OGTT) for details) within 5 minutes followed by return 
to MRI bore. Finally, paired MRI scans (PC VIPR and ASL) will be repeated over the next 60 min, with 
IV blood sampling to quantify insulin and glucose changes. MRI scan time will end approximately 60 min 
post-OGTT: 
1) for subject comfort in MRI 
2) as pilot studies indicate robust perfusion changes occur within 60 minutes 
 
ASL scans will be taken at the following time points (note times are approximate due to variance in scan 
times): 
• 
Baseline (pre-OGTT) 
• 
10 minutes after OGTT 
• 
25 minutes after OGTT 
• 
40 minutes after OGTT 
• 
55 minutes after OGTT 
 
PC VIPR scans will be taken at the following time points (note times are approximate due to variance in 
scan times): 
• 
Baseline (pre-OGTT) 
• 
15 minutes after OGTT 
• 
30 minutes after OGTT 
• 
45 minutes after OGTT 
• 
60 minutes after OGTT 

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4.3.4. Details of Procedures 
 
4.3.4.a. Informed Consent/Assent 
 
Informed consent/assent will be obtained from each participant outlining the potential risks. 
Consent/assent will be obtained at the initial screening visit and will take place prior to any other 
procedures being performed. If written consent/assent is refused, the subject will no longer be considered 
for the study. 
 
4.3.4.b. Fasting, and Caffeine, NSAID and Exercise Restriction 
 
Each subject will be asked to: 
• 
fast for a minimum of 4 hours 
• 
refrain from using NSAIDs (e.g. Ibuprofen) for a minimum of 24 hours 
• 
refrain from caffeine for a minimum of 24 hours 
• 
refrain from vigorous exercise for a minimum of 24 hours 
• 
inform the study team if any albuterol, inhaled steroids, or systemic steroids were taken within 48 
hours 
4.3.4.c. MRI Screening Document 
 
Each subject will be screened for MRI safety first during the pre-screening process (see section 4.3.1 
Pre-Screening Subjects). At the initial screening visit, the subject will fill out an MRI screening document 
provided by the research team to ensure their safety and comfort in the MRI scanner. 
 
 
4.3.4.d. Tanner Stage Questionnaire 
 
Each subject will complete a sex-specific pubertal self-assessment questionnaire to assess pubertal 
development (Tanner Stage; 46). Subjects will complete this in private without parental assistance and 
will be asked to complete the assessment from memory and to avoid examining themselves. 
 
4.3.4.e. Maximal Exercise Test 
 
 
Each subject will complete a maximal exercise test on an upright cycle ergometer. Subjects will be fitted 
with a mask or mouthpiece to measure oxygen consumption (VO2) and carbon dioxide production (VCO2), 
and a 3-lead electrocardiogram (ECG) to measure heart rate. Following a 2-minute warm-up at 60-70 

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revolutions per minute (RPM) at 25 Watts, exercise intensity will increase 25 Watts every 2 minutes until 
the subject can no longer maintain RPM≥55 for more than 5 seconds despite strong verbal 
encouragement. Average duration of maximal exercise testing in adolescents is approximately 8-12 
minutes (45, 48). 
 
 
4.3.4.f. Cognitive Tests 
 
Table 1 details the cognitive domains being assessed, the specific cognitive abilities, and the tests being 
performed. 
 
Table 1. Neuropsychological Evaluation. 
Domain 
Ability 
Tests 
Intelligence 
Verbal Functioning 
WASI (Verbal IQ) 
Intelligence 
Nonverbal Functioning 
WASI (Performance IQ) 
Memory 
Verbal memory 
 
NIH Toolbox (List Learning) 
 
Speed Function 
Psychomotor speed 
NIH Toolbox (Oral Symbol Digit Test) 
 
Executive 
Function 
Inattentiveness/inhibition 
NIH Toolbox (Flanker Inhibitory Control and Attention) 
Speed Function 
Psychomotor speed 
NIH Toolbox (Pattern Completion) 
Memory 
Visual memory 
NIH Toolbox (Picture Sequence) 
Memory 
Visual memory 
WRAML (Picture Memory) 
 
Executive  
Function 
Problem solving 
 
D-KEFS (Color-Word Interference) 
 
Executive  
Function 
Divided attention 
D-KEFS (Trail Making Test) 
Psychiatric 
Quality of Life 
Social-Emotional 
Functioning 
PedsQL - (Child 8-12/Teen 13-18) 
 
Psychiatric 
Quality of Life 
Social-Emotional 
Functioning 
Parents: 
-CBCL (Child 6-18) 
-BRIEF-II (Child 6-18) 
-PedsQL (Child 8-12/Teen 13-18) 
 
These tests will be administered by trained personnel and/or pediatric neuropsychologists via computer, 
paper forms, and interview as appropriate. 
 
4.3.4.g. Magnetic Resonance Imaging (MRI) 
 
Images will be acquired on a GE 750 3T MRI system located in the WIMR or the Waisman Center. The 
scanner is located in the WIMR within the UW Department of Radiology or at the Waisman Center 
Imaging Core. The research team will be administering the scans, as well as processing and analysis of 

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the data. No contrast agent will be used. While in the scanner, subjects will be monitored for vital signs 
(see section 4.3.4.m Subject Monitoring). All MR scanners currently used at the University of Wisconsin 
stay within FDA guidelines and have received FDA approval for use in humans. All pulse sequences 
used in this study are designed to stay within the current guidelines for dB/dt established by the FDA.  
 
The software and head coil used in this study are not intended for use in the diagnosis or treatment of 
subjects, nor are they being assessed for safety or efficacy as part of this study. Study data will not be 
submitted to the Food and Drug Administration (FDA) in support of labeling changes for these devices. 
 
PC VIPR will be utilized to acquire volumetric data sets with three-directional velocity encoding and high 
spatial resolution in fairly short scan times. This technique has been developed over the last 10 years in 
the Departments of Medical Physics and Radiology, and has been used and validated in several hundred 
subjects and have been used with regularity by the medical imaging researchers collaborating on this 
IRB proposal (Oliver Wieben). We will also conduct ASL perfusion scans. These methods are well-
characterized and used routinely during collaborative studies between Drs. Oliver Wieben and Schrage. 
 
4.3.4.h. Oral Glucose Tolerance Test (OGTT) 
 
OGTTs are clinically safe and a minimally invasive procedure to quantify IR. After a minimum 4-hour fast, 
subjects consume, within 5 min, a beverage containing 75 grams glucose (similar to a can of soda). This 
is the standard administration of OGTT (Poomthavorn, Nantarakchaikul, Mahachoklertwattana, 
Chailurkit, & Khlairit, 2013). Blood sampling occurs for 60 minutes (see section 4.3.4.j Blood Sampling). 
The Schrage lab has conducted multiple OGTT studies in IR adults and found the OGTT with MRI 
measures is well tolerated. 
 
4.3.4.i. Venipuncture 
 
Only during screening visit. Venipuncture will usually occur in the antecubital fossa or hand by trained 
personnel using standard aseptic technique.  
 
4.3.4.j. Intravenous Catheter 
 
Only during MRI-OGTT visit. An IV catheter will be placed by trained personnel in the antecubital fossa 
or hand using standard aseptic technique. Study staff trained and delegated in phlebotomy/IV placement, 
WIMR staff, research nurse, or higher study-associated clinical authority (e.g. physician) will insert an IV 
using standard medical procedures. The IV catheter will remain in place for approximately 2 hours. 
 
4.3.4.k. Blood sampling 
 
Screening visit: 
Approximately 20 mL of blood will be obtained for blood chemistry values. 
 
OGTT-MRI study visit: 
Blood draws of approximately 10-15 mL per draw to a cumulative maximum of 100 mL will be obtained 
via the IV catheter at specific time points to measure blood chemistry values (e.g. insulin, glucose). The 
specific time points are as follows:  
• 
Baseline 
• 
5 minutes after OGTT  
• 
10 minutes after OGTT  
• 
20 minutes after OGTT 

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• 
30 minutes after OGTT  
• 
45 minutes after OGTT  
• 
60 minutes after OGTT 
 
4.3.4.l. Subject Monitoring 
 
During the screening visit, specifically the maximal exercise test, subjects will be monitored for: 
• 
Heart rate (via electrocardiogram or heart rate monitor) 
• 
Oxygen consumption (via mouthpiece or face mask) 
• 
Adverse signs and symptoms 
 
Throughout the OGTT-MRI visit, subjects will be monitored for: 
• 
Blood glucose (via glucometer) 
• 
Heart rate (via electrocardiogram or pulse oximeter) 
• 
Blood pressure (via automated brachial artery auscultation) 
• 
Blood oxygen saturation (via pulse oximeter) 
• 
End-tidal carbon dioxide (via nasal cannula or mask) 
• 
Adverse signs and symptoms 
 
4.3.4.m DEXA Scan 
 
This is for comparison of body composition (amount of body fat and bone density) between subjects. The 
scanner is a clinical scanner like many hospitals have for measuring body fat and bone density and is 
operated by a trained researcher. 
 
4.3.5 Summary of Study Visits and Procedures 
 
 
Screening Visit 
Cognitive Testing Visit 1-hour MRI Visit OGTT-MRI Visit 
Fasting 
X 
X 
X 
X 
Questionnaires 
X 
X 
 
 
Body Measurements 
X 
 
 
 
Blood Draw/IV 
X 
 
 
X 
DEXA Scan 
X 
X (if needed) 
X (if needed) 
X (if needed) 
Exercise Test 
X 
X (if needed) 
X (if needed) 
X (if needed) 
Cognitive Tests 
 
X 
 
 
MRI Scans 
 
 
X 
X 
OGTT 
 
 
 
X 
Vital Signs 
X 
 
X 
X 
 
4.4. Confidentiality Protections 
 
Risks to confidentiality will be minimized by keeping copies of the documents linking study assignment 
number and the participant’s unique identifiers with the participant’s informed consent in locked cabinets 
of the offices of Dr. Bill Schrage. Only subject numbers will be used for group assignment, data 
processing, and analyses. All data will be stored in locked cabinets in a secure lab, and electronic files 
are all stored on password-protected databases and computers. 
 

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Personal information such as name, gender, date of birth, and medication history will be stored in a locked 
file cabinet in a locked office in the PI’s laboratory. Subject information will be coded to remove any 
personal identifiers during data analysis or research publications. Research oversight and regulatory 
groups may review study records. 
 
Please see section 7.9.4. Protecting the Confidentiality of Participant Data for more details. 
 
4.5. Remuneration 
 
Subjects will be paid in the following manner: 
• 
Up to $60 for the screening visit 
o $20 for completion of the virtual screening procedures 
o $10 for the blood draw 
o $10 for the DEXA scan 
o $20 for the maximal exercise test 
• 
$60-$90 for the cognitive visit, prorated at $30/hr rounded to the nearest half hour 
• 
$30 for the 1-hour MRI visit, prorated at $30/hr rounded to the nearest half hour 
• 
$60 for the OGTT-MRI visit, prorated at $30/hr rounded to the nearest half hour 
The total compensation is estimated to be $210-$240 for completing all study visits. Payment will be 
given to the participant following each visit or sent to the subject’s provided address. 
 
 

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5. BANKING AND SHARING OF DATA AND BIOSPECIMENS 
5.1. Data 
 
Blood analysis results and MRI images will be stored for future research use. Banked data will be stored 
on secure computer servers that are password-protected.  
5.2. Biospecimens 
 
Blood samples will be stored for future research use. Samples will be stored in temperature controlled 
locations (e.g. freezer) in a secure location within the Department of Kinesiology.  
5.3. Assurances 
 
Any data or samples shared with personnel outside of the study team will be coded with a study ID 
number. Unless the staff is listed as key personnel on this study, they will not be given access to the key 
linking subject identifiers with their data, samples, or study ID number. The study PI will be responsible 
for the oversight of the data and sample banking, and will review all requests to utilize the data and 
samples. The PI will be responsible for confirming that IRB approval or exemption has been granted prior 
to the release of any data or samples. Future study results obtained from banked data or samples will 
not be reported to subjects. 
 
As new information about insulin resistance or brain health becomes known, researchers may want to go 
back and analyze existing data or samples with a different aim. Additionally, combining similar information 
from multiple studies into a larger set could potentially provide more meaningful information than what 
was collected in any one study. All new analyses that do not fall under this project’s aims or that combine 
more than one dataset will be approved through the IRB as separate protocols. 
 
 

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6. RISKS ASSOCIATED WITH PROCEDURES 
 
6.1 Risk Summary 
 
This protocol is modestly invasive with only one (1) IV catheter for blood sampling, therefore overall risk 
appears modest for IV catheter and mild for OGTT and MRI. 
 
6.2 Detailed Risks 
 
6.2.1. Fasting, and Caffeine, NSAID and Exercise Restriction 
 
Risks include: 
• 
feelings of hunger 
• 
irritability 
• 
fatigue 
• 
light-headedness 
• 
dizziness 
 
There are no risks of NSAID restriction if NSAIDs are not prescribed by a physician for clinical care. There 
are no risks of acute abstention from exercise. These risks are considered minimal. See Letters of 
Support from physicians who regularly require fasting procedures in pediatric populations (section 11. 
Supplemental Information). 
 
6.2.2. Maximal Exercise Test 
 
Risks include: 
• 
Feelings of exertion, fatigue, and breathlessness 
• 
Discomfort due to VO2 monitoring equipment (i.e. mouthpiece or mask) 
• 
Serious 
complications 
including 
orthopedic 
injury, 
myocardial 
infarction, 
arrhythmia, 
hemodynamic instability, and death are rare (Takken 2016; van Brussel 2019) 
These risks are considered minimal, resolving quickly (seconds to minutes) after test termination. See 
Letters of Support from pediatric physicians who regularly perform maximal exercise testing in pediatric 
populations (section 11. Supplemental Information). 
 
6.2.3. Intravenous Catheter and Venipuncture 
 
Risks include: 
• 
bruise or clot formation 
• 
infection 
• 
pain at the site of catheter insertion/venipuncture 
• 
hematoma after withdrawal 
• 
soreness over the site  
 
These should all be transient and resolve after several days. 
 

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6.2.4. Blood sampling 
 
Risks include: 
• 
Pain at site of blood draw 
• 
Bleeding 
• 
Infection 
• 
Dizziness 
 
Poses no further risk than IV catheterization. 
 
6.2.5. OGTT 
 
Risks include: 
• 
Nausea 
• 
Vomiting 
• 
Abdominal bloating 
• 
Headache 
• 
Hypoglycemia 
 
These risks are uncommon or rare. 
 
6.2.6. MRI 
 
MRI uses a powerful magnetic field, not ionizing radiation, to create an image of the scanned area of the 
body. No contrast agent will be used. 
 
Risks include: 
• 
There are no known risks of MRI, aside from the standard risks associated with persons with 
certain metallic implants. If potential participants have a metallic implant, cardiac pacemaker, or 
are pregnant, they will be disqualified.  
• 
Sensation of claustrophobia or anxiety, particularly in individuals susceptible to it. 
• 
People with metallic implants, such as prostheses or aneurysm clips, or persons with electronic 
implants, such as cardiac pacemakers, the magnetic field generated by the magnetic resonance 
machine can cause a displacement or malfunctioning of these devices. 
• 
We know of no risks or adverse effects from the radio signals used in this study. 
• 
Some people have also reported tingling or tapping sensations, or muscle twitches in different 
parts of their body during the imaging procedure. 
o These sensations are not hazardous and should not cause discomfort to the participants.  
• 
A small increase in risk may be associated with rapid gradient waveform switching times 
associated with fast MR imaging. In certain situations, the rapid switching of gradient waveforms 
has caused peripheral nerve stimulation in subjects. 
o Significant nerve stimulation, however, has not occurred as long as the imaging system 
has been programmed to stay within certain limitations of gradient strength and switching 
time (dB/dt). 
• 
Occasionally, people who have clasped their hands tightly together during the study have reported 
a feeling of electrical shock in their hands and arms. 

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o This is also not hazardous; however, to avoid any possible discomfort, participants will be 
instructed to not clasp their hands together during the study. 
• 
Women who are pregnant must not participate in this study. The potential risks to a fetus from the 
MRI scan are not definitely known.  
• 
The MR scanner produces loud tapping sounds during operation, which may reach somewhat 
objectionable levels. 
• 
Anatomical abnormalities 
o Images will not be reviewed by neuroradiology 
o Any obvious abnormalities will be reported to the IRB upon discovery for assistance in 
determining how to proceed with potential reporting 
 
All MR scanners currently used at the University of Wisconsin stay within these guidelines and have 
received FDA approval for use in humans. All pulse sequences used in this study are designed to stay 
within the current guidelines for dB/dt established by the FDA. 
 
6.2.7. Subject Monitoring 
 
An automatic blood pressure cuff around the upper arm may feel uncomfortable while inflated, but this is 
temporary (30-60 seconds). Blood pressure measures are considered very safe.  
 
6.2.8. Cognitive Testing 
 
Sensitive information may be collected during the cognitive testing visit during the structured parental 
interview assessing the child’s quality of life. The one measure with questions that may indicate illegal 
behaviors and suicidal ideation is the Child Behavior Checklist (CBCL). 
  
The following items on the CBCL are notable: 
• 
Item 2: drinks alcohol without parental approval 
• 
Item 18: Deliberately harms self or attempts suicide 
• 
Item 28: breaks rules at home, school, or elsewhere 
• 
Item 57: physically attacks people 
• 
Item 59: plays with sex parts in public 
• 
Item 82: steals outside of the home 
• 
Item 91: talks about killing self 
• 
Item 99: smokes, chews, or sniffs tobacco 
• 
Item 101: truancy, skips school 
• 
Item 105: uses drugs for nonmedical purposes 
• 
Item 106: vandalism 
The answers to these questions may indicate illegal behaviors or psychiatric distress (e.g. suicidal 
ideation) which may be socially stigmatizing. Answers to sensitive questions will be safeguarded (see 
section 7.9.4 Protecting the Confidentiality of Participant Data). 
 
6.2.9. DEXA Scan 
 
The x-ray exposure during this test is very small (~30% of a typical chest X-ray) and is well below the 
levels that result in a high risk of harmful effects. The radiation dose for a whole-body scan is 
approximately 7 microSieverts. Using a background radiation dose of 2.4 mSv, this equates to a BERT 
value of about 24 hours (1 day). 
 

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7. DATA AND SAFETY MONITORING PLAN 
 
7.1. Monitoring study safety 
 
The study team and PI will monitor study data and safety through two different mechanisms: 
1. During study visits, trained Schrage lab staff will be present to address any immediate safety 
concerns. Schrage lab staff will document all expected and unanticipated problems as described 
in this protocol. 
2. We will complete internal audits of all subject files on a quarterly basis (see section 7.8 Internal 
Audits). These internal audits will identify any unanticipated outcomes or trends, and Schrage lab 
staff can then notify the IRB and the DMC of any new, common, or unanticipated adverse events.   
 
The PI and the research team will consult if there are subject complaints or an adverse event is 
experienced (anticipated or unanticipated). The PI and the research team will review the data to ensure 
the subject populations are safely handling the study conditions and experimental conditions, that all 
procedures are being followed per IRB approved protocol, and that all forms are completed to ensure 
compliance with IRB regulations. Further, study staff will discuss concerns or subject safety issues, 
including the potential need to modify study procedures to reduce risk.  
 
In addition to the monitoring of the study by the PI and study team, the PI has contracted with the Institute 
for Clinical and Translational Research Data Monitoring Committee (ICTR DMC) to provide independent, 
ongoing data and safety monitoring. 
 
7.2. Data Monitoring Committee (DMC) 
 
The ICTR DMC provides investigators with independent services to ensure appropriate measures are in 
place to promote subject safety, research integrity and compliance with federal regulations and local 
policies for individual clinical research protocols in need of DMC review as determined by the PI, the 
funding agency, the local Scientific Review Committee, or the local IRB, and for which no DMC exists. 
For these studies, the UW ICTR DMC will be the primary data and safety advisory group for the PI.  
 
The DMC is supported in its mission of safety and compliance by experienced ICTR staff to provide 
administrative assistance, experienced members representing a diversity of backgrounds, skills and 
knowledge, and the use of REDCap which allows more efficient tracking of protocols and protocol 
subjects. In providing oversight for the conduct of this study, the ICTR DMC will meet on a biannual basis 
throughout the study. Additional meetings may be scheduled as determined by the DMC or as requested 
by the PI. The DMC members will review protocol-specific reports created by statisticians that serve a 
non-voting member role on the DMC using data pulled from the REDCap. These standard reports will 
include an overview of study objectives, a review of actual and projected accrual rates, an evaluation of 
patient demographics for balance of randomization (if applicable), and a summary of the number and 
seriousness of adverse events. An interim analysis of study results may be performed and source 
documents may be reviewed to allow the DMC to independently judge whether the overall integrity and 
conduct of the protocol remain acceptable based on data provided and reported by the PI. The DMC will 
make recommendations to the PI that could include actions of continuation, modification, suspension, or 
termination. 
 
 

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Data Type 
Frequency of Review 
Reviewers 
Subject accrual (including compliance 
with protocol enrollment criteria) 
Every 6 months 
ICTR Data Monitoring Committee, 
PI 
Subject demographics 
Every 6 months 
ICTR Data Monitoring Committee, 
PI 
Status of all enrolled subjects as of date 
of reporting 
Every 6 months 
ICTR Data Monitoring Committee, 
PI 
Primary outcome analysis 
Every 6 months 
ICTR Data Monitoring Committee 
AEs and rates 
Every 6 months 
ICTR Data Monitoring Committee, 
PI 
SAEs 
Per occurrence 
ICTR Data Monitoring Committee, 
PI 
 
7.3. Adverse Event (AE) Reporting 
 
This study will utilize the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 for adverse 
event monitoring and reporting. This information can be downloaded from the CTEP home page 
(http://ctep.info.nih.gov/CTC4/ctc_ind_term.htm). 
 
The severity of the event will be graded using the CTCAE. In addition, for comparison to the CTCAE, 
adverse events will be tabulated using a 3-level schema as defined below: 
 
• 
Mild 
o Event may be noticeable to patient 
o Does not influence daily activities 
o Usually does not require intervention. 
 
• 
Moderate 
o Event may be of sufficient severity to make patient uncomfortable 
o Performance of daily activities may be influenced 
o Intervention may be needed. 
 
• 
Severe 
o Event may cause severe discomfort 
o Usually interferes with daily activities; patient may not be able to continue in the study 
o Treatment or other intervention usually needed.  
 
Note the distinction between the severity and the seriousness of an AE. A severe event is not necessarily 
an SAE. For example, a headache may be severe (i.e. interferes significantly with patient's usual function) 
but would not be classified as serious unless it met one of the criteria for SAEs. 
 
Next, it will be determined by the PI and study physician or clinical neuropsychologists whether the event 
is expected or unexpected and if the AE is related to the intervention or study procedures. With this 
information, it will be determined whether an AE should be reported as an expedited report in addition to 
submission via routine clinical data. 
 

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Expedited AE reporting may require e-mail notification, phone, or fax submission of a written report as 
directed below. All expedited AE reports will be submitted to the IRB of record, the UW Health Sciences 
Institutional Review Board (HS IRB) in the case of this study.   
 
7.3.1. Assessment of Attribution 
 
Attribution of AEs will be assessed in relation to the study procedures. When assessing whether an 
adverse event is related to a study procedure, the following attribution categories are utilized: 
 
• 
Definitely Related 
o An AE categorized as definite is clearly related to study procedures. If the timing of the AE 
is definitely consistent with the exposure to the study related procedures and it is most 
likely that the AE was caused by the study procedures such as because a high occurrence 
of the AE was expected based on the study intervention materials. In this case, the PI may 
categorize the AE as definitely related. 
 
• 
Probably Related 
o An AE that is likely related to study procedures. If the timing of the AE is consistent with 
the exposure to the study intervention and it is more likely that the AE was caused by the 
study procedures than not, the PI may categorize the AE as Probable. 
 
• 
Possibly Related 
o An AE that may be related to study procedure. If the timing of the AE is reasonably 
consistent with the exposure to the study intervention, and there is another cause of the 
AE that could be equally likely, the PI may categorize the AE as Possible. 
 
• 
Unlikely Related 
o An Unlikely AE is one that is doubtfully related to study procedures. The coincidence of 
the AE with the exposure of the investigational product or intervention should be assessed. 
An AE that continued while the intervention was interrupted or stopped, or if the AE 
resolved while the intervention continued, may be categorized as Unlikely. If there is 
another more likely cause of the AE, the PI may determine that the AE was unlikely related 
to the study intervention. 
 
• 
Not Related/Unrelated 
o An Unrelated AE is one that is clearly NOT related to study procedures. An AE may be 
considered Unrelated if the subject did not receive the study intervention or if there is 
another obvious cause of the AE (for example, a car accident or other disease/condition). 
 
7.4. Serious Adverse Event (SAE) 
 
A serious adverse event (SAE) is an AE occurring during any phase of the study (i.e., screening, 
admission, treatment, or follow-up) that fulfils one or more of the following criteria: 
• 
Results in death 
• 
Is immediately life-threatening 
• 
Requires in-patient hospitalization or prolongation of existing hospitalization (more than 24 hours) 

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• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital abnormality or birth defect 
• 
Is an important medical event that may jeopardize the subject or may require medical intervention 
to prevent one of the outcomes listed above 
 
 

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7.5. Identifying, Reviewing, and Reporting Adverse Events and Unanticipated Problems to the IRB 
 
Identifying 
• 
To effectively identify all AEs (anticipated or unanticipated), Schrage lab staff will follow laboratory 
and protocol specific SOPs, complete AE reporting forms for each subject as they arise, and 
maintain a database of all reported adverse events for each protocol by entering all AEs into 
REDCap within a timely manner. All stopping events or subject reported complaints/concerns will 
be documented via study notes during the study. Participants will be encouraged to contact us 
with any questions or concerns following the study visit.  
 
Reviewing 
• 
The PI, study physician, and study team will also review AEs during internal audits of study 
documents in a cumulative fashion to identify any trends of safety concerns (see section 7.8 
Internal Audits). In the event of an SAE, the PI, study physician, DMC, and IRB will review the 
event as soon as possible. 
 
Reporting 
• 
All adverse events will be recorded in REDCap in a timely manner consistent with reporting 
requirements. Schrage lab staff will log all AEs in REDCap, and report AEs and unanticipated 
problems to the DMC and to the IRB as indicated by IRB guidance on Knowledge Base.  
 
7.6. Expedited Adverse Event Reporting Requirements 
 
Serious Adverse Event— Reported Within 24 Hours 
• 
Serious Adverse Events requiring expedited reporting within 24 hours will be reported to both the 
UW Health Sciences IRB and the ICTR DMC Manager within one working day as directed below. 
Confirmation that all appropriate parties were notified will be done at this time.   
 
Serious Adverse Event – Reported within 15 Days 
• 
Serious Adverse Events requiring expedited reports in writing within 15 working days will be sent 
by the PI and study team. 
 
7.7. Protocol Deviations 
 
To effectively identify deviations from the IRB approved protocol, Schrage Lab staff will maintain protocol 
checklists that are designed to not only minimize protocol deviations but to identify deviations when they 
do occur. Additionally, Schrage Lab staff will conduct regular internal audits (see section 7.8 Internal 
Audits) to identify deviations that may have been overlooked. All deviations will be logged on a Deviation 
Tracking Log and a database of deviations will be maintained in REDCap. The PI will review deviations 
as they are identified, and oversight committees will be notified according to their guidelines. The study 
team will implement corrective action plans when appropriate to address deviations. 
 
7.8. Internal Audits 
 
Regular internal audits will be conducted to ensure compliance and safety. Internal audits will occur on a 
quarterly basis. These audits could consist of full or partial review of study records, depending on 

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observed past compliance. Audits will be documented on Internal Compliance Review forms modified 
specifically for the study and will include review of the following: 
• 
Personnel (e.g., ensure all personnel have the appropriate training) 
• 
Documentation of IRB submissions and correspondence 
• 
Informed Consent/HIPAA Authorization Forms 
• 
Laboratory Aspects of the Trial 
• 
Equipment Maintenance 
• 
Communication and Correspondence with oversight committees 
• 
Study Conduct 
• 
Completion of Case Report Forms 
• 
Adverse Events 
• 
Protocol deviations 
These internal audits will identify any unanticipated outcomes or trends, and Schrage Lab staff can then 
notify the IRB of any new, common, or unanticipated adverse events, if applicable. 
 
The results from internal audits (e.g., noncompliance, adverse event trends) will be discussed during 
regular lab meetings where team members can establish plans to prevent future issues. 
 
7.9. Minimizing Research-Associated Risk 
 
First and foremost, risks will be minimized by the prescreening procedures to determine preliminary 
eligibility, which includes thorough health history and MRI safety questionnaires (see section 4.3.1. Pre-
Screening Subjects) which will be verified by the subject’s parent/guardian (if <18 years of age). 
Secondly, in-person anthropometric measurements and survey of pregnancy (see section 4.3.2. In-
Person Screening) will further establish safety for subject participation. This two-stage process should 
minimize risk prior to any study conditions being imposed on the participants. 
 
Once enrolled as a subject and participation in the trials begins, we will monitor heart rate, blood oxygen 
saturation, blood pressure, end-tidal carbon dioxide, and subject comfort throughout the study visit (if 
applicable; see section 4.3.4.m. Subject Monitoring). This data will be used to monitor the health of our 
participants and for research purposes.  
 
Having only trained personnel performing the IV catheter placement under aseptic conditions will 
minimize IV catheter placement risk. 
 
Screening each subject for cardiovascular disease and any blood clotting disorders will minimize 
intravenous catheter placement risk. 
 
If a participant reports positive pregnancy, the study team will immediately end further screening 
procedures and will only state that the subject is not eligible without details as to the reasoning. This is 
done to protect the confidentiality of the pregnancy screening procedure (see section 4.3.2. In-Person 
Screening). 
 
Although we are extensively screening out subjects that have overt diabetes, the results of the 
experimental OGTT may produce results identifying that a subject may be diabetic which may indicate 
a clinical concern. The fact that insulin analysis will occur towards the end of the study (due to cost 
limitations with assay) means diabetic subjects will have completed all Aims of study. A diabetes finding 
does not pose further risk to the patient. 
 

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Using the standard exclusionary procedures to prevent subjects with hazardous metallic implants, cardiac 
pacemakers, or subjects who are pregnant will minimize the risks of MRI scanning. A standard safety 
screening form used for clinical MRI scanning will be used to exclude patients with hazardous metallic 
implants, cardiac pacemakers, or patients who are pregnant. In addition, all operators of the MRI 
equipment are trained in the proper use of hearing protection, padding, and subject monitoring. To 
minimize any discomfort, participants will be provided with disposable earplugs or headphones to wear 
during the procedure. These earplugs will protect their ears but still allow communication with the 
technician running the scan. 
 
With constant monitoring by the study team under the supervision of Drs. Schrage and Carrel, we will 
record and identify trends and report any significant problems to the IRB. We will compile an electronic 
record of any adverse events associated with each subject and will review these events as they occur. 
We will also review cumulative events to identify trends as well as approaches to stop trends and 
approach the IRB with appropriate protocol modifications.  
 
7.9.1. Initial screening 
 
All subjects will be asked to complete an initial pre-screening, documenting physical activity, medical 
history, medications, and MRI safety (see section 4.3.1. Pre-Screening Subjects). The questions are 
designed to immediately eliminate those subjects meeting exclusion criteria. A detailed initial screening 
script and survey is included in the formal IRB proposal. Potential subjects who are ineligible, or who 
decline to participate, will have their screening information deleted or destroyed. 
 
During the in-person screening, if the subject is ostensibly ineligible (e.g. weight<94.5 lbs), we will stop 
the screening process immediately. The reason for stoppage will be documented. This will not be 
considered a deviation to the protocol. 
 
Other studies in the PI’s lab investigate the same subject population and utilize similar screening 
procedures. A subject recruited from another study can have their screening information from that study 
used for the purposes of this study to minimize subject burden. Any procedures that were not included in 
the other studies’ screening process that are required for this study will be conducted. Screening 
information will be valid for 180 days. 
 
7.9.2. Subject Monitoring 
 
Testing may include subject monitoring of heart rate, oxygen saturation, end tidal carbon dioxide, and 
blood pressure (if applicable; see section 4.3.4.m. Subject Monitoring). With continuous monitoring, we 
can readily observe any adverse events in real time and move to stop procedures immediately and return 
to baseline conditions. In case of serious cardiovascular event, a defibrillator is located nearby all testing 
areas, and CPR certified staff is present during procedures. In the case of emergency, 911 will be 
contacted. Further, in the event of a “code” for procedures at the WIMR, the WIMR is now part of the 
UWHC Code response area such that emergency team response time is excellent. 
 
7.9.3. Blood Sampling 
 
Blood sampling (not venipuncture or IV placement) will be performed only by research personnel that 
have had phlebotomy training. This training has encompassed the insertion, maintenance, and use of 
intravenous catheters. These personnel all have at least a bachelor’s degree or higher health and/or 
physiology-related education in terms of academic preparation. We feel education and training of 

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personnel, and our positive track record over 10 years at UW performing similar invasive procedures, 
provide high confidence IV blood draws present a very low risk to subjects.  
 
7.9.4. Protecting the Confidentiality of Participant Data 
 
Risks to confidentiality will be minimized by keeping copies of the documents linking study assignment 
number and the participant’s unique identifiers with the participant’s informed consent in locked cabinets 
of the offices of Dr. Bill Schrage. Only subject numbers will be used for group assignment, data 
processing, and analyses. All data will be stored in locked cabinets in a secure lab, and electronic files 
are all stored on password-protected databases and computers. 
 
Personal information such as name, gender, date of birth, and medication history will be stored in a locked 
file cabinet in a locked office in the PI’s laboratory. Subject information will be coded to remove any 
personal identifiers during data analysis or research publications. Research oversight and regulatory 
groups may review study records. 
 
Data will be stored as both hard copies and electronic files. Hard copy data collection forms will be locked 
in a file cabinet in the restricted-access laboratory area or in the Department of Neurology (cognitive 
testing files only). Electronic files that contain potential Protected Health Information (PHI) will be stored 
in REDCap, a secure web-based data management system, and/or a secure Box folder configured with 
the involvement of the UW-Madison HIPAA security officer. Electronic files will be HIPAA compliant and 
will use a subject ID instead of the subject’s name. The raw MRI data will be stored on servers assigned 
to Dr. Wieben and managed by the Departments of Medical Physics and Radiology and/or on encrypted 
external hard drives. 
 
Blood samples may be analyzed for insulin and inflammatory markers externally to the study team (e.g. 
UW Primate Research Center). Samples analyzed at the UW Health Clinical Labs will be placed in the 
medical record of the subjects per UW Health policy. Samples analyzed at research labs (e.g. UW 
Primate Research Center) will remain coded such that the outside blood analysis team will not be 
provided any identifiers or means by which they could connect the blood samples to any individuals that 
have participated in the study. 
 
7.10. Stopping Guidelines 
 
7.10.1. Study Stopping Guidelines 
 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause. Written notification, documenting the reason for study suspension or termination, will be provided 
by the suspending or terminating party to investigator, funding agency, and regulatory authorities. If the 
study is prematurely terminated or suspended, the PI will promptly inform the IRB and will provide the 
reason(s) for the termination or suspension. Circumstances that may warrant termination or suspension 
include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to subjects 
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable  
The study may resume once concerns about safety, protocol compliance, and data quality are addressed 
and satisfy the applicable regulatory authorities. 
 

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7.10.2 Subject Stopping Guidelines 
 
Each intervention (i.e. study visit) will be terminated based on the following specific guidelines: 
• 
If the subject wishes to stop 
• 
If the subject complains of serious pain or discomfort 
• 
If the subject is admitted to hospital for psychiatric distress 
• 
If the subject complains of (related to MRI): 
o Discomfort 
o Anxiety 
o Claustrophobia 
• 
If baseline glucose is ≥126 mg/dL and is not the result of non-compliance with fasting requirement 
(see 4.1.2. Exclusion Criteria) 
• 
Any SAE occurring during study procedures 
 
 

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8. STATISTICAL CONSIDERATIONS 
 
Covariates for all analyses include sex, age, adiposity, aerobic fitness, and education unless noted 
otherwise. 
 
The overarching statistical approach uses multiple regression to determine whether IR is inversely 
associated with outcome measures in Aims 1 and 2 and to assess mediation in Aim 3. Mean/summed 
outcomes (total cognition, total CBF or CP) will be predicted by HOMA-IR, for example. Not only is a 
composite measure calculated as a mean more reliable than the subdomain scores, but averaging 
multiple measures of a single construct yields a more powerful hypothesis test than using variables 
individually (40), under conditions we expect to hold here. This approach avoids inflation of Type I error 
rate as each hypothesis test examines the relationship among single variables or composites, not among 
multiple subdomain measures. If the test of the mean or composite score is significant, then there is no 
increased Type I error rate incurred in subsequent subdomain/regional tests (41). 
 
8.1. MRI Data Processing 
  
Standard and validated tools will be used for image processing. The research team is expert in 
analyzing brain imaging data and have published several studies focused on MRI-derived blood flow-
based measures.  
  
Maps representing gray matter probability and cerebral blood flow will be generated using SPM, FSL, 
or similar software. Scans will be processed using the most up to date and recommended processing 
scheme, including methods previously used by our research group and collaborators.  
  
We expect to use standard viewing software (Advanced Workstation 4.3, GE Healthcare) for magnetic 
resonance angiography and phase contrast data, in addition to 
1) MIMICS and EnSight, commercial software packages commonly used in 4D flow visualization and 
2) MatLab-based software for quantitative analysis of 4D data sets 
 
Quantitative data from two 4D multi venc flow acquisitions will be available. The data includes: 
1) Quantitative blood flow values (peak and mean flow, peak and mean velocity, pulsatility and 
resistance index) of 4D PC 
2) Quantitative morphological data (vessel diameter) of:  
a. Standard magnetic resonance angiography techniques 
b. 4D PC 
 
8.2. Aim 1 
 
Composite scores will be computed by calculating and averaging z-scores within each cognitive domain. 
Composite total cognitive scores will be predicted from HOMA-IR and the covariates. Pilot data yielded 
a partial correlation of –0.6 between HOMA-IR and the total cognitive score. A one-tail test of the 
population correlation coefficient with a Type I error rate of 0.05 requires a sample size of N=13 to yield 
power of 0.80. If this composite score test is significant, similar analyses will be performed to examine 
the relationship between HOMA-IR and individual cognitive ability subdomain scores. 
 
 
 

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8.3. Aim 2 
 
8.3.1. Aim 2 Data Expression 
 
For conceptual clarity, pilot data are expressed as CBF or CP. For statistics, the key dependent variable 
in Aim 2 is cerebrovascular conductance (CVC), where CVC is CBF or CP normalized to mean arterial 
pressure (i.e. blood pressure), where 
 
CVC=CBF/mean arterial pressure 
 
CVC is critical, as: 
 
1) it normalizes CBF/CP for differences in mean arterial pressure (up to 15 mmHg across IR spectrum) 
2) it accounts for mean arterial pressure changes with OGTT 
3) changes in CVC (∆CVC) have a predictable relationship independent of baseline differences, where 
+∆CVC indicates dilation and –∆CVC indicates constriction (24, 53) 
CVCMACRO refers to macrocirculation (i.e. CBF) and CVCMICRO to microcirculation (i.e. CP). While 
CVCMACRO and CVCMICRO are physiologically related, the two levels are conceptually independent. Testing 
both is crucial as preliminary data indicate resting CBF is normal, yet CP is lower as IR increases. 
 
8.3.2. Aim 2A 
 
Total CVC will be predicted from HOMA-IR and the covariates (intracranial volume is an additional 
covariate for CVC analyses). Resting CP data indicate reduced perfusion in several anterior brain 
regions. Pilot data yielded a correlation between HOMA-IR and resting CVCMICRO of –0.324, such that a 
sample of size N=55 will yield the power of 0.8 in a one-tailed test of the correlation, with Type I error rate 
of 0.05. In contrast, we will test, but do not expect, resting CVCMACRO to be related to HOMA-IR, since 
resting CVCMACRO is similar in low and high IR. 
 
8.3.3. Aim 2B 
 
The change in CVC (∆CVC) will be predicted from HOMA-IR and the covariates, where  
 
∆CVC=(peak CVC during OGTT)-(resting CVC) 
 
Pilot data indicate the average correlation between HOMA-IR and ∆CVCMICRO of -0.42, so a one-tailed 
test of the partial correlation will yield power of 0.80 with a sample size of N=31, with Type I error rate of 
0.05. We plan to assess numerous regions of interest (ROIs) linked to executive functions and memory, 
including frontal, temporal, and parietal regions (e.g. middle, medial, and superior frontal gyri, anterior 
cingulate, hippocampus, middle and superior temporal gyri, posterior cingulate, precuneus), as well as 
ROIs with less involvement in memory or executive functions (e.g. precentral and postcentral gyri, 
occipital cortex). ∆CVCMICRO is only predicted from HOMA-IR within an ROI, not between, to limit the 
number of comparisons and therefore limit the false discovery rate. 
 
For ∆CVCMACRO, up to 11 extra- and intra cranial arteries will be studied (basilar, 2 ACA, 2 MCA, 2 PCA, 
2 vertebral, and 2 ICA; dependent on individual anatomy). Total CBF will be calculated, where 
 
CBFTOTAL = CBFBASILAR + CBFICA-Right + CBFICA-LEFT 
 

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Pilot data indicate that the average correlation between HOMA-IR and MCA perfusion during OGTT is -
0.796, so that a one-tailed test of the partial correlation will yield the power of 0.80 with a sample size 
N=6, with Type I error rate of 0.05. For all of Aim 2, if either total CVC test is significant (micro or macro), 
similar regression analyses will be performed to examine the mediating effects of ∆CVC between HOMA-
IR and individual ROI (micro) or artery (macro) ∆CVC values. 
 
8.4. Aim 3 
 
Aim 3 tests the mediation effects of CVC on the relationship between HOMA-IR and the composite 
cognition scores assessed in Aim 1. Mediation will be assessed using the test of joint significance (23). 
The path between HOMA-IR and perfusion will have been examined in Aim 2. At rest (Aim 3A), mean 
CVCMICRO and CVCMACRO will be examined as the primary mediating variables. Pilot data indicate the 
correlation between resting CVCMICRO and cognition is 0.37, so that a sample size of N=41 would yield 
0.8 power in a one-tailed test and Type I error rate of 0.05. During OGTT (Aim 3B), mean peak ∆CVC 
will be the primary mediator variable. Pilot data indicate that the partial correlation between post-OGTT 
∆CVCMICRO and the total cognitive score was 0.4, so that a directional test of this path requires a sample 
size of N=34 to yield the power of 0.8 with Type I error rate of 0.05. A similar power is achieved for 
CVCMACRO. If either test (rest or OGTT challenge) is significant, analyses will be performed to examine 
the mediating effects of CVC between HOMA-IR and individual cognitive ability domain scores. 
 
8.5. Statistical Summary 
 
With N=55 for the overall grant, power in Aim 1 increases to near unity, power in Aim 2A is 0.8, power in 
Aim 2B increases to 0.95, power in Aim 3A increases to 0.9, and that in Aim 3B increases to 0.94, 
providing this study with sufficient to outstanding power to test the hypotheses of interest. 

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9. DATA AND RECORD KEEPING 
 
9.1. Data Management 
 
Delegated and appropriately trained study team members will be involved in data collection. All personnel 
who will be involved in data collection have completed appropriate Human Subjects Protection training 
or will before they engage in contact with subjects and subject data. 
 
The Research Electronic Data Capture (REDCap) system is used to manage the data for this study.  
REDCap is a largely self-service, secure, web-based application for building and managing data 
collection forms. REDCap provides data management functionality by allowing the development of 
instruments and surveys to support data capture for research studies.  
 
The REDCAP system used for this study is managed by ICTR. The ICTR REDCap support team will 
work with the investigator, statistician, and study team to ensure the relevant, applicable study data are 
collected and managed with restricted access using the software electronic data collection form 
instruments. 
 
9.2. Assured Confidentiality 
 
The raw MRI data will be stored on servers assigned to Dr. Wieben and managed by the Departments of 
Medical Physics and Radiology. The UW-Madison School of Education houses the Schrage Lab server 
and all electronic files will be coded to maintain HIPPA compliance. Subject information and study 
tracking information will also be stored in REDCap, which is a secure site used for data management. 
 
Any forms with PHI will be stored in locked cabinets inside the PI’s secure laboratory. All data collected 
electronically will be password protected. Only coded data will be used in data analysis. Any publications 
arising from this protocol will not include any personal identifying information or study code, thereby 
making the reported data completely de-identified. 
 
9.3. Data Collection Methods 
 
Data will be collected using REDCap, MRI, offline data collection software (e.g. LabChart), data analysis 
software, and data collection forms. These files will consist of electronic DICOM, .dat, LabChart, and 
Excel files, as well as hard copies of data collection forms for each subject. 
 
All other study data referenced above will be collected using study visit checklists and data collection 
forms, most of which constitute both the Case Report Form (CRF) and the original source (i.e., the first 
and only place the data is manually written/recorded). These study specific forms will be developed and 
maintained with the assistance of the ICTR DMC for recording all necessary data for each subject. It is 
the PI’s responsibility to ensure that these are properly, legibly, and fully completed and signed where 
appropriate. The CRFs are used to record study data and are an integral part of the study and subsequent 
reports. Therefore, the CRFs must be completed for each subject screened or enrolled according to the 
subject’s source data on a per-visit basis. All study visit checklist and data collection forms will be retained 
in the subject research chart/file. All applicable data collected using the study visit checklists and data 
collection forms will be entered in to electronic Case Report Forms (eCRFs) within REDCap. 
 
When the study is complete, the applicable CRFs must be signed by the investigator to attest that it is an 
accurate and complete record. 
 

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9.4. Retention of Study Records 
 
In compliance with UW-Madison regulations, study records will be for a minimum retained for seven years 
following final project close-out. 
 
 
 

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10. DATA INTEGRITY 
 
Integrity of data is vital for successful completion of the study. To that end, we will consider data to be 
valid if: 
• 
all cognitive testing is performed 
• 
three (3) of five (5) ASL scans are performed on the OGTT-MRI visit 
• 
three (3) of seven (7) blood draws are performed on the OGTT-MRI visit 
• 
maximal exercise testing meets a minimum of 2 of the following criteria: 
o plateau in oxygen uptake evidenced by <2 mL/kg/min increase in the last 60 seconds of 
the test 
o heart rate ≥90% of age-predicted maximal heart rate (i.e. 182-187 beats per minute) (45, 
48) 
o respiratory exchange ratio (VCO2:VO2) ≥1.0 (45, 48) 
 
These criteria are chosen based on the specific aims of the study (see section 3. Specific Aims and 
Preliminary Evidence) and for outcome measures to be appropriately assessed (see section 8. Statistical 
Considerations). Any procedures not performed will be documented with an explanation as to why it was 
not performed. Protocol deviations will only be filed if the aforementioned data are not collected or valid. 
 
We will invite subjects to return to complete the specific testing that was not valid as stated previously if 
the reason for not obtaining that data was not due to reaching a stopping guideline (see section 7.10. 
Stopping Guidelines). The number of re-invitations to perform a specific procedure will be limited to one 
(1) to reduce repeated exposure to procedures. Such instances will likely be due to: 
• 
technical difficulties which temporarily impede data collection 
• 
delays and subsequent time constraints 
• 
failure of attaining a valid maximal exercise testing response 
Regarding the maximal exercise testing, on re-invitation, subjects will only need to undergo the maximal 
exercise test but not the other screening procedures performed previously. 
 
Having this flexibility will allow the study team to attempt the data collection again on a subject that has 
experienced other procedures (i.e. may be farther along in the study) without needing to expose a new 
subject to these procedures. This will hopefully limit the number of subjects experiencing these 
procedures and assist the study team in attaining the target sample size. 
 
We will continue enrollment until our target sample size (see section 4.1 Subject Population) is studied 
in full. 
 
 

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11. REFERENCES 
 
1. Andersen SL. Trajectories of brain development: point of vulnerability or window of opportunity? 
Neuroscience & Biobehavioral Reviews 27: 3–18, 2003. 
 
2. Arsenijevic Y, Weiss S. Insulin-like growth factor-I is a differentiation factor for postmitotic CNS stem 
cell-derived neuronal precursors: distinct actions from those of brain-derived neurotrophic factor. J 
Neurosci 18: 2118–2128, 1998. 
 
3. Baker LD, Cross DJ, Minoshima S, Belongia D, Watson GS, Craft S. Insulin resistance and 
Alzheimer-like reductions in regional cerebral glucose metabolism for cognitively normal adults with 
prediabetes or early type 2 diabetes. Arch Neurol 68: 51–57, 2011. 
 
4. Birdsill AC, Carlsson CM, Willette AA, Okonkwo OC, Johnson SC, Xu G, Oh JM, Gallagher CL, 
Koscik RL, Jonaitis EM, Hermann BP, LaRue A, Rowley HA, Asthana S, Sager MA, Bendlin BB. 
Low cerebral blood flow is associated with lower memory function in metabolic syndrome. Obesity 21: 
1313–1320, 2013. 
 
5. Blakemore S-J, Choudhury S. Development of the adolescent brain: implications for executive function 
and social cognition. Journal of Child Psychology and Psychiatry 47: 296–312, 2006. 
 
6. Blakemore S-J. Imaging brain development: The adolescent brain. NeuroImage 61: 397–406, 2012. 
 
7. Carro E, Trejo JL, Busiguina S, Torres-Aleman I. Circulating insulin-like growth factor I mediates the 
protective effects of physical exercise against brain insults of different etiology and anatomy. J Neurosci 
21: 5678–5684, 2001. 
 
8. Cholerton B, Baker LD, Craft S. Insulin, cognition, and dementia. Eur J Pharmacol 719: 170–179, 
2013. 
 
9. Craft S, Baker LD, Montine TJ, Minoshima S, Watson GS, Claxton A, Arbuckle M, Callaghan M, 
Tsai E, Plymate SR, Green PS, Leverenz J, Cross D, Gerton B. Intranasal insulin therapy for 
Alzheimer disease and amnestic mild cognitive impairment: a pilot clinical trial. Arch Neurol 69: 29–38, 
2012. 
 
10. Craft S. The role of metabolic disorders in Alzheimer disease and vascular dementia: two roads 
converged. Arch Neurol 66: 300–305, 2009. 
 
11. Dai W, Garcia D, de Bazelaire C, Alsop DC. Continuous flow-driven inversion for arterial spin labeling 
using pulsed radio frequency and gradient fields. Magn Reson Med 60: 1488–1497, 2008. 
 
12. FORD ES, LI C, ZHAO G. Prevalence and correlates of metabolic syndrome based on a harmonious 
definition among adults in the US*. Journal of Diabetes 2: 180–193, 2010. 
 
13. Garcia DM, Duhamel G, Alsop DC. Efficiency of inversion pulses for background suppressed arterial 
spin labeling. Magn Reson Med 54: 366–372, 2005. 
 
14. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, Dai S, Ford ES, Fox CS, 
Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Huffman MD, Judd SE, 
Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Mackey RH, Magid DJ, Marcus 
GM, Marelli A, Matchar DB, McGuire DK, Mohler ER, Moy CS, Mussolino ME, Neumar RW, Nichol 

Insulin Resistance, Cognitive Health, and Perfusion of the Adolescent Brain 
Version: 9 
Version Date: 09.24.2021 
43
G, Pandey DK, Paynter NP, Reeves MJ, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Wong 
ND, Woo D, Turner MB, on behalf of the American Heart Association Statistics Committee and 
Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics--2014 Update: A Report From the 
American Heart Association. Circulation 129: e28–e292, 2014. 
 
15. Hales CM, Carroll MD, Fryar CD, Ogden CL. Prevalence of Obesity Among Adults and Youth: United 
States, 2015-2016. NCHS Data Brief : 1–8, 2017. 
 
16. Hoscheidt SM, Kellawan JM, Berman SE, Rivera-Rivera LA, Krause RA, Oh JM, Beeri MS, Rowley 
HA, Wieben O, Carlsson CM, Asthana S, Johnson SC, Schrage WG, Bendlin BB. Insulin resistance is 
associated with lower arterial blood flow and reduced cortical perfusion in cognitively asymptomatic 
middle-aged adults. Journal of Cerebral Blood Flow & Metabolism 37: 2249–2261, 2016. 
 
17. Hoscheidt SM, Starks EJ, Oh JM, Zetterberg H, Blennow K, Krause RA, Gleason CE, Puglielli L, 
Atwood CS, Carlsson CM, Asthana S, Johnson SC, Bendlin BB. Insulin Resistance is Associated with 
Increased Levels of Cerebrospinal Fluid Biomarkers of Alzheimer's Disease and Reduced Memory 
Function in At-Risk Healthy Middle-Aged Adults. J Alzheimers Dis 52: 1373–1383, 2016. 
 
18. Hu Q, Lee SY, O'Kusky JR, Ye P. Signalling through the type 1 insulin-like growth factor receptor 
(IGF1R) interacts with canonical Wnt signalling to promote neural proliferation in developing brain. ASN 
Neuro 4: AN20120009, 2012. 
 
19. Iadecola C. The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in 
Health and Disease. Neuron 96: 17–42, 2017. 
 
20. Katakam PV, Snipes JA, Steed MM, Busija DW. Insulin Induced Uncoupling of Nitric Oxide Synthase 
and Generation of Reactive Oxygen Species Underlie Cerebrovascular Insulin Resistance. Circulation 
124, 2011. 
 
21. Katakam PVG, Snipes JA, Steed MM, Busija DW. Insulin-induced generation of reactive oxygen 
species and uncoupling of nitric oxide synthase underlie the cerebrovascular insulin resistance in obese 
rats. J Cereb Blood Flow Metab 32: 792–804, 2012. 
 
22. Kleinridders A, Ferris HA, Cai W, Kahn CR. Insulin action in brain regulates systemic metabolism and 
brain function. Diabetes 63: 2232–2243, 2014. 
 
23. Krause MR, Serlin RC, Ward SE, Rony RYZ, Ezenwa MO, Naab F. Testing mediation in nursing 
research: beyond Baron and Kenny. Nurs Res 59: 288–294, 2010. 
 
24. Lautt WW. Resistance or conductance for expression of arterial vascular tone. Microvascular 
Research 37: 230–236, 1989. 
 
25. Laws SM, Gaskin S, Woodfield A, Srikanth V, Bruce D, Fraser PE, Porter T, Newsholme P, 
Wijesekara N, Burnham S, Dore V, Li Q-X, Maruff P, Masters CL, Rainey-Smith S, Rowe CC, 
Salvado O, Villemagne VL, Martins RN, Verdile G. Insulin resistance is associated with reductions in 
specific cognitive domains and increases in CSF tau in cognitively normal adults. Sci Rep 7, 2017. 
 
26. Lezak MD, Howieson DB, Bigler ED, Tranel D. Neuropsychological Assessment. Oxford University 
Press, 2012. 
 
27. Limberg JK, Kellawan JM, Harrell JW, Johansson R, Eldridge MW, Proctor LT, Sebranek JJ, 

Insulin Resistance, Cognitive Health, and Perfusion of the Adolescent Brain 
Version: 9 
Version Date: 09.24.2021 
44
Schrage WG. Exercise-mediated vasodilation in human obesity and metabolic syndrome: Effect of acute 
ascorbic 
acid 
infusion. 
Am. 
J. 
Physiol. 
Heart 
Circ. 
Physiol. 
(July 
18, 
2014). 
doi: 
10.1152/ajpheart.00312.2014. 
 
28. Mikhail Kellawan J, Harrell JW, Roldan-Alzate A, Wieben O, Schrage WG. Regional hypoxic cerebral 
vasodilation facilitated by diameter changes primarily in anterior versus posterior circulation. 
Journal of Cerebral Blood Flow &amp; Metabolism 37: 2025–2034, 2017. 
 
29. Mikhail Kellawan J, Harrell JW, Schrauben EM, Hoffman CA, Roldan-Alzate A, Schrage WG, 
Wieben O. Quantitative cerebrovascular 4D flow MRI at rest and during hypercapnia challenge. Magn 
Reson Imaging 34: 422–428, 2016. 
 
30. Narasimhan S, Weinstock RS. Youth-Onset Type 2 Diabetes Mellitus: Lessons Learned From the 
TODAY Study. Mayo Clin Proc 89: 806–816, 2014. 
 
31. Neumann KF, Rojo L, Navarrete LP, Farías G, Reyes P, Maccioni RB. Insulin resistance and 
Alzheimer's disease: molecular links & clinical implications. Curr Alzheimer Res 5: 438–447, 2008. 
 
32. O'Kusky J, Ye P. Neurodevelopmental effects of insulin-like growth factor signaling. Front 
Neuroendocrinol 33: 230–251, 2012. 
 
33. Ogden CL, Carroll MD, Lawman HG, Fryar CD, Kruszon-Moran D, Kit BK, Flegal KM. Trends in 
Obesity Prevalence Among Children and Adolescents in the United States, 1988-1994 Through 2013- 
2014. JAMA 315: 2292–2299, 2016. 
 
34. Okonkwo OC, Xu G, Oh JM, Dowling NM, Carlsson CM, Gallagher CL, Birdsill AC, Palotti M, 
Wharton W, Hermann BP, LaRue A, Bendlin BB, Rowley HA, Asthana S, Sager MA, Johnson SC. 
Cerebral blood flow is diminished in asymptomatic middle-aged adults with maternal history of 
Alzheimer's disease. Cereb Cortex 24: 978–988, 2014. 
 
35. Olver TD, Grunewald ZI, Jurrissen TJ, MacPherson RE, LeBlanc PJ, Schnurbusch TR, Czajkowski 
AM, Laughlin MH, Rector RS, Bender SB, Walters EM, Emter CA, Padilla J. Microvascular insulin 
resistance in skeletal muscle and brain occurs early in the development of juvenile obesity in pigs. Am J 
Physiol Regul Integr Comp Physiol 13: ajpregu002132017, 2017. 
 
36. Pate, R. R., Ross, R., Dowda, M., Trost, S. G., & Sirard, J. R. (2003). Validation of a 3-day physical 
activity recall instrument in female youth. Pediatric exercise science, 15(3), 257-265. 
 
37. Rodriguez-Colon SM, Mo J, Duan Y, Liu J, Caulfield JE, Jin X, Liao D. Metabolic syndrome clusters 
and the risk of incident stroke: the atherosclerosis risk in communities (ARIC) study. Stroke 40: 200– 205, 
2009. 
 
38. Roger VL, Go AS, Lloyd-Jones DM, Adams RJ, Berry JD, Brown TM, Carnethon MR, Dai S, de 
Simone G, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Greenlund KJ, Hailpern SM, Heit JA, Ho 
PM, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Makuc DM, 
Marcus GM, Marelli A, Matchar DB, McDermott MM, Meigs JB, Moy CS, Mozaffarian D, Mussolino 
ME, Nichol G, Paynter NP, Rosamond WD, Sorlie PD, Stafford RS, Turan TN, Turner MB, Wong 
ND, Wylie-Rosett J, on behalf of the American Heart Association Statistics Committee and Stroke 
Statistics Writing Group. Heart Disease and Stroke Statistics--2011 Update: A Report From the 
American Heart Association. Circulation 123: e18–e209, 2011. 
 

Insulin Resistance, Cognitive Health, and Perfusion of the Adolescent Brain 
Version: 9 
Version Date: 09.24.2021 
45
39. Rusinek H, Ha J, Yau PL, Storey P, Tirsi A, Tsui WH, Frosch O, Azova S, Convit A. Cerebral 
perfusion in insulin resistance and type 2 diabetes. Journal of Cerebral Blood Flow &amp; Metabolism 
(October 15, 2014). doi: 10.1038/jcbfm.2014.173. 
 
40. Schilling TM, Ferreira de Sá DS, Westerhausen R, Strelzyk F, Larra MF, Hallschmid M, Savaskan E, 
Oitzl MS, Busch H-P, Naumann E, Schächinger H. Intranasal insulin increases regional cerebral blood 
flow in the insular cortex in men independently of cortisol manipulation. Hum Brain Mapp 35: 1944–1956, 
2014. 
 
41. Serlin RC. An empirical comparison of three methods for performing univariate analyses with 
multivariate data. Montreal, Quebec, Canada: 1999. 
 
42. Serlin RC. Fisher Was Right. Journal of Modern Applied Statistical Methods 9: 2–8, 2010. 
 
43. Strauss E, Sherman EMS, Spreen O. A Compendium of Neuropsychological Tests. Oxford University 
Press, 2006. 
 
44. Sweat V, Yates KF, Migliaccio R, Convit A. Obese Adolescents Show Reduced Cognitive Processing 
Speed Compared with Healthy Weight Peers. Child Obes 13: 190–196, 2017. 
 
45. Takken, T., Bongers, B. C., Van Brussel, M., Haapala, E. A., & Hulzebos, E. H. (2017). 
Cardiopulmonary exercise testing in pediatrics. Annals of the American Thoracic 
Society, 14(Supplement 1), S123-S128. 
 
46. Taylor, S. J., Whincup, P. H., Hindmarsh, P. C., Lampe, F., Odoki, K., & Cook, D. G. (2001). 
Performance of a new pubertal self-assessment questionnaire: a preliminary study. Paediatric and 
perinatal epidemiology, 15(1), 88-94. 
 
47. Thambisetty M, Beason-Held LL, An Y, Kraut M, Metter J, Egan J, Ferrucci L, O'Brien R, Resnick 
SM. Impaired glucose tolerance in midlife and longitudinal changes in brain function during aging. 
Neurobiol Aging 34: 2271–2276, 2013. 
 
48. Van Brussel, M., Bongers, B. C., Hulzebos, E. H., Burghard, M., & Takken, T. (2019). A Systematic 
Approach to Interpreting the Cardiopulmonary Exercise Test in Pediatrics. Pediatric exercise science, 
(00), 1-10. 
 
49. Weston, A. T., Petosa, R., & Pate, R. R. (1997). Validation of an instrument for measurement of 
physical activity in youth. Medicine & Science in Sports & Exercise. 
 
50. Willette AA, Bendlin BB, Starks EJ, Birdsill AC, Johnson SC, Christian BT, Okonkwo OC, La Rue A, 
Hermann BP, Koscik RL, Jonaitis EM, Sager MA, Asthana S. Association of Insulin Resistance With 
Cerebral Glucose Uptake in Late Middle-Aged Adults at Risk for Alzheimer Disease. JAMA Neurology 
72: 1013–1020, 2015. 
 
51. Willette AA, Burns CM, Bendlin BB. Insulin Resistance and APOEε4. JAMA Neurology 72: 1536–
1537, 2015. 
 
52. Xu G, Rowley HA, Wu G, Alsop DC, Shankaranarayanan A, Dowling M, Christian BT, Oakes TR, 
Johnson SC. Reliability and precision of pseudo-continuous arterial spin labeling perfusion MRI on 3.0 T 
and comparison with 15O-water PET in elderly subjects at risk for Alzheimer's disease. NMR Biomed 23: 
286–293, 2010. 

Insulin Resistance, Cognitive Health, and Perfusion of the Adolescent Brain 
Version: 9 
Version Date: 09.24.2021 
46
53. Yates KF, Sweat V, Yau PL, Turchiano MM, Convit A. Impact of Metabolic Syndrome on Cognition 
and Brain: A Selected Review of the Literature. Arteriosclerosis, Thrombosis, and Vascular Biology 32: 
2060–2067, 2012. 
 
54. Yau PL, Castro MG, Tagani A, Tsui WH, Convit A. Obesity and metabolic syndrome and functional 
and structural brain impairments in adolescence. PEDIATRICS 130: e856–64, 2012. 
 
55. Yau PL, Javier DC, Ryan CM, Tsui WH, Ardekani BA, Ten S, Convit A. Preliminary evidence for 
brain complications in obese adolescents with type 2 diabetes mellitus. Diabetologia 53: 2298–2306, 
2010. 
 
56. Yau PL, Kang EH, Javier DC, Convit A. Preliminary evidence of cognitive and brain abnormalities in 
uncomplicated adolescent obesity. Obesity 22: 1865–1871, 2014. 
 
57. Ye FQ, Frank JA, Weinberger DR, McLaughlin AC. Noise reduction in 3D perfusion imaging by 
attenuating the static signal in arterial spin tagging (ASSIST). Magn Reson Med 44: 92–100, 2000. 
 
58. The paradox of sympathetic vasoconstriction in exercising skeletal muscle. [Online]. 29: 159–163, 
2001. 
http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=11688788&retmode=ref&cmd=
prlinks. 
 
59. Reduced glucose tolerance is associated with poor memory performance and hippocampal atrophy 
among normal elderly. 100: 2019–2022, 2003. 
 
60. PC VIPR: a high-speed 3D phase-contrast method for flow quantification and high-resolution 
angiography. 
[Online]. 
26: 
743–749, 
2005. 
http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=15814915&retmode=ref&cmd=
prlinks. 
 
61. Improved 3D phase contrast MRI with off-resonance corrected dual echo VIPR. 60: 1329–1336, 2008. 
 
62. Cerebrovascular responses to insulin in rats. 29: 1955–1967, 2009. 
 
63. Age and the metabolic syndrome as risk factors for ischemic stroke: improving preclinical models of 
ischemic 
stroke. 
[Online]. 
85: 
523–539, 
2012. 
http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=23239952&retmode=ref&cmd=
prlinks. 
 
 
 
 
 
 
 

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