Sympathetic Activation in Obesity
Phase: NA
Conditions: Obesity, Healthy
Sponsor: Yale University

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CLINICAL STUDY PROTOCOL
Interventional Drug or Biologic
Sympathetic activation in obesity
Protocol Number
2000030749
Protocol Version
03/14/2022
Version #2
PRINCIPAL INVESTIGATOR:
Name Renata Belfort De Aguiar
Department Internal Medicine / Section of Endocrinology 
Telephone Number 203-785-2440
Email Address Renata.aguiar@yale.edu
IND Number:
IND Holder:
NCT Number: NCT04946552
Study Phase (1, 2, 3, or 4-postmarketing):
Confidentiality Statement:
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REVISION HISTORY:
Revision #
Version Date
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Synopsis
Primary Objective
The primary objective of this study is to determine whether sympathetic nervous system (SNS) 
activity in adipose tissue (WAT and BAT), muscle and brain is altered in individuals with obesity in 
comparison to individuals with normal weight. Simultaneous multi-organ SNS activation will be 
obtained with a radiotracer for norepinephrine transporters (NET) for whole-body Positron 
Emission Tomography in combination with low dose computed tomography (PET/CT) imaging and 
microneurography (gold standard test for assessment of muscle SNS activity).
Secondary Objective (if applicable)
The secondary objectives of this study are: 1) to evaluate whether gender differences affect 
peripheral SNS in healthy normal weight and obese men and women in adipose tissue (WAT and 
BAT) and muscle (resting/fasting); 2) To investigate the relationship between peripheral and 
central SNS activity in obesity, by correlating SNS activity in peripheral tissues (WAT, BAT, and 
muscle) and brain; and 3) To investigate NET CNS and peripheral SNS activity before and after a 
high carbohydrate mixed meal in lean and obese men and women.
Study Duration 
4 years
Study Design
Aim: To measure peripheral SNS in healthy normal weight and obese men and women in 
adipose tissue (WAT and BAT), deltoid skeletal muscle, and brain NET at fast and after a 
mixed meal test (MMT).
The goal of this study is to measure in 20 individuals with normal weight (BMI 18-25 kg/m2) and 20 
individuals with obesity (BMI 30-50 kg/m2) NET-binding in WAT and BAT to better understand the 
role of SNS in obesity. SNS activation of a) peripheral tissues: adipose tissue (BAT and WAT) and 
skeletal muscle; and b) CNS NET will be measure synchronously by using [11C]MRB whole body 
PET/CT imaging in combination with the gold standard measurement of peripheral SNS activity 
(muscle sympathetic nervous activity (MSNA)).
Visit 1 - Screening procedures:
Potential subjects will complete pre-screening over the telephone to determine preliminary eligibility 
based on inclusion/exclusion criteria and basic socio-demographic information will be collected. 
Potentially qualifying participants will then be screened in person at the Yale New Haven Hospital 
(YNHH) Research Unit (HRU) or Church Street Research Unit (CSRU) with vital signs, EKG, a 
medical history and physical exam. Fasting blood will be collected for HbA1C, creatinine, liver 
enzymes (ALT, AST), TSH, hematocrit, and lipid panel. A urine pregnancy test will be administered.
Oral glucose tolerance test (OGTT) will be administered as follows: Following a 10-hour overnight 
fast, a nurse will insert an intravenous (IV) catheter. Subsequently, subjects will ingest 7.5 oz of 
Glucola, which contains 75 g of dextrose in orange flavored water. Blood samples will be taken at -
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15, 0, 10, 20, 30, 60, 90 and 120 minutes (after glucola ingestion) for the measurement of plasma 
glucose, insulin, and C-peptide concentrations, and calculation of measurements of insulin 
sensitivity index (Matsuda Index). Approximately 40 mL of blood will be drawn at this visit.
Body Composition, Percent Body Fat & Percent Body Water: will be assessed using bioelectrical 
impedance analysis Tanita® scale (FDA cleared); which is a special multi frequency seg-mental 
body composition analyzer that delivers a very mild electrical current that allows measurement of 
fat mass, percent body fat, fat free mass, total body water, and percent body water. This test will be 
performed at screening visit. For better accuracy, measurement of percent fat and percent body 
water will be obtained with whole body Dual X-Ray Absorptiometry (DXA) scan (Hologic®) (located 
in the HRU) on screening-OGTT visit. The scanner arm will move over the participant’s body from 
feet to head. The machine uses a small amount of radiation (one tenth of the amount of radiation 
from a chest x-ray) to measure body fat, muscle and bone density.
Visit 2 – Whole body PET/CT Imaging Visit - FASTING:
Two weeks after the screening visit, participants who qualified for the study will be asked to come 
to the PET Center to undergo the whole body [11C]MRB PET scan. Participants will be asked to 
maintain their regular exercise routine.
Human PET-CT Protocol: [11C]MRB will be synthesized at the Yale PET center based on procedures 
described and previously performed 1,2. An intravenous bolus dose of MRB (~20mCi) will be injected 
by an infusion pump. PET data will be acquired dynamically for 120 min using the Siemens mCT- 
X whole-body PET/CT scanner using continuous bed motion to image perform multiple PET bed 
acquisitions from the top of the head to the lower abdominal region. A CT scan will be performed 
for attenuation correction and to help delineate the BAT and other regions of interest (ROIs). Images 
will be reconstructed with an ordered subset expectation maximization algorithm using point spread 
function correction and time-of-flight information. To delineate fat (WAT, BAT) and skeletal muscle 
regions of interests (ROIs), CT images are first resliced to match the resolution and location of PET 
images. As performed in previous studies 2, a supraclavicular ROI is first manually drawn on the CT 
images. Within that area, the fat ROI is segmented using the CT images and an intensity window 
from -200 to -50 HU. Subsequently the fat ROI is segmented into WAT and BAT ROIs by using the 
11C-MRB PET images: Standardized Uptake Value (SUV) ≥ 1.25 for BAT, SUV < 1.25 for WAT. 
Brain ROIs for predetermined NET-rich areas of the brain, including the locus ceruleus, raphe, 
thalamus and hypothalamus were defined in template space and are applied to the PET images by 
using the participant’s MR image as an intermediate step. For analysis of tracer uptake, mean SUV 
will be reported for segmentation of adipose tissue. Because the distribution volume ratio (DVR) is 
a more precise measurement of uptake for reversible tracers 3 such as 11C-MRB, regional 
quantification of 11C-MRB uptake as DVR (VT/ VND), estimated via multilinear reference tissue model 
2 (MRTM2) 4,5 (t*=30 min) using the occipital cortex, a region with low NET density 2,5,6 as the 
reference region. MRTM2 reduces the variability of MRTM (4) parameter estimates by fixing one of 
the three parameters, the parameter k2', which is only related to the reference region time-activity 
curve, to a common value for all target regions. In this study, the parameter k2' was fixed to 0.021 
min-1, based on previous brain studies and previous BAT PET studies 2. In addition to DVR, MRTM2
can estimate R1, the relative radioligand delivery, using a 1 tissue compartment model (1TC). For
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the 1TC model, volume of distribution, VT = K1/k2, where K1 (mL/min−1/mL−1) is the rate constant of 
radiotracer from plasma to tissue of target organ and k2 is the rate constant from tissue to plasma. 
Using a reference region with non-displaceable binding (e.g. occipital lobe) we can estimate similar 
rate constants: VND = K’1/k’2. Recalling that K1 and K’1 are the rate constants for transfer from plasma 
to the tissue and reference region, respectively, then we can estimate relative radioligand delivery 
as R1 = K1/K’1.
Visit 3 – Whole body PET/CT Imaging Visit - High-Carbohydrate Mixed Meal Test (MMT)
High Carbohydrate Mixed meal test (MMT): Two weeks after visit 2, participants will come to the 
PET Center after a 10-hour overnight fast. A nurse will insert an intravenous (IV) catheter and 
subjects will ingest a liquid meal (65% CHO, 20% fat, and 15% protein, equal to 40% of daily energy 
expenditure, per ~16 ounces) to be prepared by the Metabolic Kitchen located at the HRU. Blood 
samples will be collected at −10, 0, 15, 30, 60, 90, and 120 min) for measurement of glucose, insulin 
and C-peptide concentrations 7. Plasma samples will be drawn at baseline and throughout the MMT 
for measurement of additional hormones (such as leptin, ghrelin, thyroid hormones, and 
catecholamines) and free fatty acids (FFA) levels. Insulin and glucose will be used to calculate 
indexes of insulin sensitivity 7-9. Hormones, glucose and FFA will be correlated with PET 
measurements of brown fat at baseline and during the MMT. Approximately 60 mL of blood will be 
drawn at this visit.
Participants will undergo whole body [11C]MRB PET scan as described in visit 3. After the MMT, 
PET data will be acquired dynamically for 120 min using the Siemens mCT-X whole-body PET/CT 
scanner after an intravenous bolus dose of MRB (~20mCi).
The order of the PET scan visits 2 and 3 will be randomized.
Measurement of Energy Intake: Hunger and fullness will be determined immediately before, during 
and after the PET scans with Visual Analog Scales (VAS).
Visit 4 – Sympathetic Nervous System Activity (SNSA) measurement Visit - Fasting
Two weeks after visit 3, participants will be asked to return for a separated visit for measurements 
of SNS activity at rest and fasting. This visit will be performed at the Pierce Laboratory (Dr. 
Stachenfeld’s laboratory). The participants will come in the morning (~9am) and will be asked to fast 
for 10-hour and to maintain their regular exercise routine. The following measurements will be 
obtained: MSNA, heart rate (HR), blood pressure (BP), electrocardiogram (EKG), and heart rate 
variability,. The proposed procedures will be performed at fasting (baseline). Participants will be 
asked to maintain their regular exercise routine.
Muscle sympathetic nerve activity (MSNA): Microneurography is the gold standard, most sensitive 
and reliable MSNA assessment in humans. The primary measure of sympathetic nervous system 
activity is peripheral nerve activity, as measured by MSNA.
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Muscle sympathetic nerve activity will be quantified by identification and measurement of 
sympathetic bursts in the integrated neurogram and expressed as burst frequency (number of bursts 
per unit time). ECG surface electrodes are applied to the chest, an elastic band incorporating a 
strain gauge element is strapped around the thorax for recording respiratory movements, and a 
sensor cuff of a servopulse-plethysmography arterial pressure monitor (FINAPRES, Ohmeda) is 
placed on the middle phalanx of the long finger (held at heart level). The experiments are carried 
out in an environmentally controlled chamber at an ambient temperature of 27.0 ± 0.1 °C. The 
experiment will begin with a 60-minute control period. During such time the subjects rest in the 
reclined position.
Measurements of HR, BP, and EKG (beat-to-beat) will also be obtained during the procedure. An 
intravenous catheter will be inserted into a forearm vein for blood sampling. Following the 
instrumentation period, resting HR, pulse interval (PI), BP, and MSNA will be recorded for 10 
minutes. Following these measurements, plasma will be taken for measurements of insulin, glucose, 
free fatty acids, norepinephrine, and epinephrine levels. Approximately 20 mL of blood will be drawn 
at this visit. After the first blood sample and the 10-min rest phase, subjects will begin a seven- 
minute period of rest with paced breathing (12 breaths per minute) while HR, BP, PI and MSNA are 
measured. The subjects will synchronize breathing to a metronome set to a cadence of ~12 breaths 
per minute (0.2 Hz).
Procedure: Microneurography Recording of multiunit postganglionic muscle sympathetic nerve 
activity (MSNA) will be made from the common peroneal nerve as it winds around the fibular head 
with the subject in the supine position with the subject’s thigh comfortably supported. This procedure 
requires us to first trace the course of the nerve using small electrical stimuli applied to the surface 
of the skin over the nerve or use the ultrasound to visually assist. This allows the microneurographer 
to find and focus the recording electrode tip on a nerve fascicle associated with muscle sympathetic 
activity. The time required to focus the electrode on the nerve averages about 30 min and will not 
exceed 60 min. In optimal conditions the track of the nerve from superficial stimulation is accurate 
and the nerve remains in position during insertion. An uninsulated subcutaneous electrode will serve 
as the reference. Mechanoreceptor afferent activity can be recorded during light tapping on the 
muscle belly or passive muscle stretch. A suitable intrafascicular recording site for MSNA consists 
of regular bursts and synchronous with the cardiac cycle. The neural activity is amplified (2x104), 
filtered (0.5-5.0 kHz), rectified, and integrated. The amplified and filtered nerve signal is also led to 
an audio monitor and through a resistance capacitance circuit (time constant 100 ms). The 
integrated nerve signal is digitized at 400 Hz along with the ECG signal while respiratory and arterial 
pressure signals are also sampled at 400 Hz (MacLab 8s). Data for analysis is selected from periods 
of quiet breathing in which subjects were relaxed and not talking. Microneurography was approved 
under HIC protocols # 8079, #0512000875, #1609018353 and is currently used under # 
2000020950.
Cardiovascular variables. Recorded with a 16-channel computerized data-acquisition system at a 
sampling Speed of 400 Hz (ADI Instruments PowerLab 9, Castle Hill, Australia). Beat-to-beat DBP, 
SBP and pulse interval (PI) are determined using peak detection algorithms (44). Brachial artery BP 
and HR are measured with an automated sphygmomanometer (Colin Medical Instruments Corp,
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Komaki, Japan). Heart rate and pulse interval will be determined beat-to-beat from lead II of the 
EKG recording.
Visit 5 – Sympathetic Nervous System Activity (SNSA) measurement Visit – Mixed Meal Test 
(MMT)
Two weeks after visit 4, participants will be asked to return for a separated visit for measurements 
of SNS activity at rest and after intake of liquid meal. Participants will undergo similar procedures 
described on visit 4. However, MSNA will be obtained after MMT. The participants will come in the 
morning (~9am) and will be asked to fast for 10-hour and to maintain their regular exercise routine. 
A nurse will insert an intravenous (IV) catheter and subjects will ingest a liquid meal (65% CHO, 
20% fat, and 15% protein, equal to 40% of daily energy expenditure, per ~16 ounces) to be prepared 
by the Metabolic Kitchen. Measurements of HR, BP, and EKG (beat-to-beat) will be obtained during 
the procedure. An intravenous catheter will be inserted into a forearm vein for blood sampling. 
Following the instrumentation period, resting HR, pulse interval (PI), BP, and MSNA will be recorded 
for 10 minutes. Following these measurements, plasma will be taken for measurements of insulin, 
glucose, free fatty acids, norepinephrine, and epinephrine levels. Approximately 40 mL of blood will 
be drawn at this visit.
Participants will be asked to maintain their regular exercise routine. The order of SNSA Fasting and 
MMT visits (visits 4 and 5) will be randomized. All study visits will be conducted within an 8-week 
period (including the two PET and SNSA scans).
.
Schematic figure of the visits and procedures to be done. The order of the PET scans will be randomized.
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Abbreviations: BP=blood pressure, EKG=electrocardiogram, HR=heart rate, MSNA=Muscle sympathetic nerve activity, 
SNSA=Sympathetic Nervous System Activity
PET and MSNA visits do not need to occur in sequence, i.e. PET scans can occur before or after the MSNA.
Number of Study Sites
1
Study Population
We plan to enroll 20 participants with normal BMI (10 women and 10 men) and 20 with obesity (10 
women and 10 men).
Number of Participants
40
Primary Outcome Variables
Primary outcomes: Group differences in 1) [11C]MRB-binding in brown adipose tissue (BAT), white 
adipose tissue (WAT), skeletal muscle, and norepinephrine-rich brain regions (hypothalamus and 
thalamus); and 2) microneurography (muscle sympathetic nerve activity (MSNA)). Correlations 
between NET in the brain and peripheral NET and MSNA.
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Secondary and Exploratory Outcome Variables (if applicable)
Secondary endpoints: Plasma hormone levels, heart rate, blood pressure, and electrocardiogram 
(beat-to-beat). Correlations of secondary endpoints with NET in the brain and peripheral NET and 
MSNA.
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Abbreviations
Explanation
Abbreviation
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Glossary of Terms
Explanation
Glossary
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Table of Contents
Synopsis ..................................................................................................................................3
Primary Objective...............................................................................................................3
Secondary Objective (if applicable)..................................................................................3
Study Duration....................................................................................................................3
4 years.................................................................................................................................3
Study Design ......................................................................................................................3
Number of Study Sites.......................................................................................................8
Study Population................................................................................................................8
Number of Participants......................................................................................................8
Primary Outcome Variables ..............................................................................................8
Secondary and Exploratory Outcome Variables (if applicable).....................................8
Abbreviations ...........................................................................................................................9
Glossary of Terms..................................................................................................................10
1
Introduction.....................................................................................................................16
1.1
Introductory Statement ............................................................................................16
2
Background.....................................................................................................................17
2.1.1
Preclinical Experience......................................................................................17
2.1.2
Clinical Experience...........................................................................................17
2.2
Background/prevalence of research topic ...............................................................21
3
Rationale/Significance ....................................................................................................23
3.1
Problem Statement..................................................................................................23
3.2
Purpose of Study/Potential Impact ..........................................................................23
3.2.1
Potential Risks..................................................................................................23
3.2.2
Potential Benefits .............................................................................................26
4
Study Objectives.............................................................................................................27
4.1
Hypothesis...............................................................................................................27
4.2
Primary Objective ....................................................................................................27
4.3
Secondary Objectives (if applicable) .......................................................................27
4.4
Exploratory Objectives (if applicable) ......................................................................27
5
Study Design ..................................................................................................................28
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5.1
General Design Description.....................................................................................28
5.1.1
Study Date Range and Duration.......................................................................32
5.1.2
Number of Study Sites .....................................................................................32
5.2
Outcome Variables..................................................................................................33
5.2.1
Primary Outcome Variables .............................................................................33
The primary objective of this study is to determine whether sympathetic nervous system 
(SNS) activity in adipose tissue (WAT and BAT), muscle and brain is altered in 
individuals with obesity in comparison to individuals with normal weight. Simultaneous 
multi-organ SNS activation will be obtained with a radiotracer for norepinephrine 
transporters (NET) for whole-body Positron Emission Tomography (PET) imaging and 
microneurography (gold standard test for assessment of muscle SNS activity).............33
Primary outcome: SNS activity measured by [11C]MRB binding in adipose tissue, muscle 
and..................................................................................................................................33
brain; and sympathetic burst in muscle (measured with microneurography)..................33
5.2.2
Secondary Outcome Variables (if applicable) ..................................................33
The secondary objectives of this study are: 1) to evaluate whether gender differences 
affect peripheral SNS in healthy normal weight and obese men and women in adipose 
tissue (WAT and BAT) and muscle (resting/fasting); 2) To investigate the relationship 
between peripheral and central SNS activity in obesity, by correlating SNS activity in 
peripheral tissues (WAT, BAT, and muscle) and brain; and 3) To investigate NET CNS 
and peripheral SNS activity before and after a high carbohydrate mixed meal in lean 
and obese men and women............................................................................................33
5.2.3
Exploratory Outcome Variables (if applicable) .................................................33
N/A..................................................................................................................................33
5.3
Study Population .....................................................................................................33
5.3.1
Number of Participants.....................................................................................33
We plan to enroll 20 participants with normal BMI (10 women and 10 men) and 20 with 
obesity (10 women and 10 men).....................................................................................33
5.3.2
Eligibility Criteria/Vulnerable Populations.........................................................34
6
Methods..........................................................................................................................34
6.1
Treatment ................................................................................................................34
6.1.1
Identity of Investigational Product ....................................................................34
6.1.2
Dosage, Administration, Schedule ...................................................................35
N/A..................................................................................................................................35
6.1.3
Method of Assignment/Randomization.............................................................35
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6.1.4
Blinding and Procedures for Unblinding ...........................................................35
6.1.5
Packaging/Labelling .........................................................................................35
N/A..................................................................................................................................35
6.1.6
Storage Conditions...........................................................................................35
N/A.........................................................................................................................................35
6.1.7
Concomitant therapy ........................................................................................35
6.1.8
Restrictions.......................................................................................................35
6.2
Assessments ...........................................................................................................35
6.2.1
Efficacy.............................................................................................................35
6.2.2
Safety and Pregnancy-related policy................................................................35
6.2.3
Adverse Events Definition and Reporting.........................................................35
6.2.4
Pharmacokinetics (if applicable).......................................................................41
6.2.5
Biomarkers (if applicable).................................................................................41
N/A..................................................................................................................................41
6.3
Study Procedures....................................................................................................41
6.3.1
Study Schedule ................................................................................................45
6.3.2
Informed Consent.............................................................................................46
6.3.3
Screening .........................................................................................................46
6.3.4
Enrollment ........................................................................................................47
6.3.5
On Study Visits.................................................................................................48
........................................................................................................................................49 
6.3.6
End of Study and Follow-up .............................................................................49
6.3.7
Removal of subjects.........................................................................................49
6.4
Statistical Method ....................................................................................................49
6.4.1
Statistical Design..............................................................................................49
6.4.2
Sample Size Considerations ............................................................................50
6.5
Planned Analyses....................................................................................................50
6.5.1
Primary Objective Analysis...............................................................................50
6.5.2
Secondary Objectives Analyses.......................................................................51
6.5.3
Exploratory Objectives Analyses (if applicable) ...............................................51
6.5.4
Safety ...............................................................................................................51
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6.5.5
Analysis of Subject Characteristics ..................................................................51
6.5.6
Interim Analysis (if applicable)..........................................................................51
6.5.7
Health economic evaluation .............................................................................51
6.5.8
Other ................................................................................................................52
6.5.9
Subsets and Covariates ...................................................................................52
6.5.10
Handling of Missing Data .................................................................................52
7
Trial Administration.........................................................................................................53
7.1
Ethical Considerations: Informed Consent/Assent and HIPAA Authorization..........53
7.2
Institutional Review Board (IRB) Review.................................................................53
7.3
Subject Confidentiality.............................................................................................53
7.4
Deviations/Unanticipated Problems.........................................................................54
7.5
Data Collection ........................................................................................................54
7.6
Data Quality Assurance...........................................................................................54
7.7
Study Records.........................................................................................................54
7.8
Access to Source Documents .................................................................................55
7.9
Data or Specimen Storage/Security ........................................................................55
7.10
Retention of Records...............................................................................................55
7.11
Study Monitoring......................................................................................................55
7.12
Data Safety Monitoring Plan....................................................................................55
After review of the safety data, SAEs and AEs confirmed to have been related to the 
radiotracer will be reported to the FDA and the appropriate regulatory committees at Yale. 
The study will be placed on hold until a determination is made as to whether the study 
may continue as written, if modifications are needed, or if the study will be discontinued.55
7.13
Study Modification ...................................................................................................55
7.14
Study Discontinuation..............................................................................................56
7.15
Study Completion ....................................................................................................56
7.16
Conflict of Interest Policy .........................................................................................56
7.17
Funding Source .......................................................................................................56
NIH/NIDDK 1R56DK129344-01 .........................................................................................56
7.18
Publication Plan.......................................................................................................56
8
Appendices.....................................................................................................................57
9
List of Tables ..................................................................................................................58
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10
References:.................................................................................................................59
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1 Introduction
1.1
Introductory Statement
This document is a protocol for a human research study. The purpose of this protocol is to 
ensure that this study is to be conducted according to ICH GCP guidelines, and according to 
CFR 21 Part 312, other applicable government regulations and Institutional research policies 
and procedures.
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2 Background
2.1.1
Preclinical Experience
Obesity is one of the main drivers of insulin resistance, diabetes, and cardiovascular disease, 
and obesity-associated dysfunction of the sympathetic nervous system (SNS) may be 
implicated in the pathophysiology of these comorbidities 15-18. The SNS is integral to 
modulating energy homeostasis via activation of target organs, such as adipose tissue, 
skeletal muscle, liver, pancreas, and adrenal glands 19. However, this multi-organ SNS 
activation has been shown to be dysfunctional in obesity 20,21. Central nervous system (CNS) 
noradrenergic neurons release norepinephrine (NE) in these target organs. NE recycling is a 
tightly regulated process modulated in large part by the norepinephrine transporter (NET) 22. 
Due to its important role in regulating NE signaling, NET has been considered a marker of 
noradrenergic system activity 22.
In humans, in vivo quantification of NET levels can be measured using a selective positron 
emission tomography (PET) radioligand for NET, (S,S)-11C-O-methylreboxetine ([11C]MRB) 
23,24, and our group has shown that [11C]MRB can measure NET in multiple tissues including 
brown (BAT) and white adipose tissue (WAT), skeletal muscle, and brain 12,25. Furthermore, 
our preliminary data using [11C]MRB scanning in healthy lean individuals as well as individuals 
with obesity suggest that obesity is associated with marked differences in NET availability in 
BAT 2 as well as the brain 26. Use of PET allows us to assess NET binding in multiple tissues 
simultaneously. In these proposed studies, we will use NET-binding to investigate the role of 
SNS dysfunction in healthy men and women with obesity. Furthermore, because there is 
strong evidence that food induced SNS activity increment is blunted in individuals with obesity 
15,27,28, we will also test the impact of obesity on response to a relevant stimulus (food). Our 
overall premise is that coordinated multi-organ response (brown and white adipose 
tissue, muscle, liver, pancreas) is modulated by the SNS both in the fasting state and 
after a meal; however, this coordinated response becomes dysfunctional in obesity. 
Therefore, to better understand the potential for dysregulated coordination of the SNS in 
obesity, we will synchronously measure SNS activation of a) peripheral tissues: adipose tissue 
(BAT and WAT) and skeletal muscle; and b) CNS by using [11C]MRB whole body PET imaging 
in combination with muscle SNS activity (MSNA) during rest both in the fasting state and after 
a meal.
2.1.2
Clinical Experience
[11C]MRB can be used to image NET-rich regions in the body at rest. Using a rodent 
model, [11C]MRB PET imaging has been shown to appropriately 1) identify NET-rich regions 
in the body including BAT, heart, liver, pancreas, and kidney, 2) NET could be used as a highly 
specific target for BAT, and 3) that BAT can be detected in rodents under both room 
temperature and cold conditions 24. Furthermore, the binding of [11C]MRB in BAT was 
completely abolished by the pretreatment with a NET inhibitor, demonstrating the specific 
binding of the proposed ligand to BAT 24. Building upon those rodent studies, the use of 
[11C]MRB for imaging human BAT was validated under both cold and room temperature (RT) 
conditions at rest 12. Ten healthy, Caucasian subjects (5M/5F: age 25±3, BMI 22±3 kg/m2)
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underwent [11C]MRB PET-CT imaging for cervical/supraclavicular BAT under RT and cold- 
stimulated conditions (RPCM Cool vest; enthalpy 15°C) compared to 18F-FDG PET-CT 
imaging. Uptake of [11C]MRB, was quantified as the distribution volume ratio (DVR). As seen 
in Figure 1, BAT [11C]MRB binding was similar under both RT and cold conditions (BAT DVR: 
RT 1.0±0.3 vs. cold 1.1±0.3, p=0.31). Importantly, BAT DVR [11C]MRB at RT correlated 
positively with body temperature (r=0.92, p=0.004).
Figure 1. A) Representative subject during scanning at RT and cold; B) [11C]MRB 
Distribution Volume Ratio (DVR) in all subjects at RT and cold, closed marker = female
Obesity is associated with marked decrease in [11C]MRB binding of human BAT. 
Recently, we have shown that there is a marked difference in [11C]MRB binding of BAT 
amongst obese compared to lean women 2 (Figure 2). In this proof of concept study, 15 healthy, 
non-diabetic Caucasian women (9 lean, age 25.6 ± 1.7, BMI 21.8 ± 1.3 kg/m2 and 6 obese age 
30.8 ± 8.8 BMI 37.9 ± 6.6 kg/m2) underwent PET-CT imaging of the neck/supraclavicular region 
using [11C]MRB under RT conditions. We observed that [11C]MRB binding was reduced in 
women with obesity in comparison to the lean group suggestive of decreased BAT. However, it 
remains to be determined whether NET in BAT is also reduced in men with obesity.
Figure 2. Decreased [11C]MRB binding in BAT in women with obesity. A) Two 
representative subjects; B) All subjects.
Fig. 3a
Figure 3a. 
Skeletal 
Muscle 
[11C]MRB DVR
correlates with 
BMI.
Obesity is associated with 
altered [11C]MRB binding
in skeletal muscle. Because SNS innervation of skeletal muscle has been shown to modulate 
skeletal muscle insulin resistance, we also performed preliminary analysis of 11CMRB binding
B
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Figure 4: Example of Muscle sympathetic nervous
activity (MSNA) in a patient with obesity and 
polycystic ovary syndrome (PCOS).
igure 6. Scatterplots correlating the changes in [ C]MRB BP
in the
ND
ypothalamus and glucose infusion rate (GIR) and counterregulatory 
ormones response to hypoglycemia.
20
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in muscle in these lean and obese women. In women, skeletal muscle (deltoid area) [11C]MRB 
DVR correlated with BMI (Figure 3a), and was marginally lower (P=0.088) in women with 
obesity compared with women with normal BMI.
Based on these initial results, our goal is to expand our understanding of the importance of 
NET-binding in peripheral tissues (BAT and WAT) and skeletal muscle in obesity which may 
play a role in the pathogenesis of obesity-associated insulin resistance, type 2 diabetes and 
hypertension. We plan to perform whole body [11C]MRB PET imaging to quantify NET-binding 
(an indicator of sympathetic activation of target organs) during rest in the fasted state in 
individuals with both normal BMI and with obesity. Furthermore, because in the past only 
women were included in our preliminary obesity study, we plan to determine whether [11C]MRB 
binding in BAT will also be decreased in men with obesity 2.
Furthermore, to validate NET as an indicator of SNS activity, we plan to correlate the results 
from the whole body [11C]MRB PET
imaging 
scans 
with 
established 
measurements of SNS, such as heart 
rate variability, plasma catecholamine 
levels and MSNA 29. Dr. Stachenfeld, one 
of the co-investigators in this project, is an 
expert in human studies of the autonomic 
nervous system. Her laboratory has used 
microneurography to measure MSNA in 
women with obesity 
30. The bottom 
tracing in Figure
4 
shows 
sympathetic 
bursts 
as 
measured by microneurography in the 
peroneal nerve of an obese human while 
resting. The other tracings show
beat to beat blood pressure, EKG and respiration. In our studies, we will examine changes in 
burst activity to determine changes in MSNA.
Human obesity is associated with altered brain NET. Central noradrenergic neuronal 
dysfunction has been observed in obesity 26,31-33. Li et al 26 demonstrated that obese subjects 
have decreased NET-availability in the thalamus (a sensory relay region in the brain); although 
these findings were not confirmed in a European cohort 34. Vettermann et al 35 showed that 
NET activity in the insula and hippocampus predicts successful weight loss. Furthermore, NET 
inhibitors used for the treatment of attention deficit disorder (ADHD) can promote weight loss 
36,37. In a prior study from our group 25, we observed that NET in the hypothalamus correlated 
with counterregulatory hormonal and metabolic response to hypoglycemia (a stimulus to 
induce stress response
and   increase    hunger) F
(Figure 6). However, it is h 
not clear whether NET- h rich 
regions in the CNS,
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such as hypothalamus, thalamus, and brain stem (areas involved with glucose metabolism 
and autonomic function) are important in regulating SNS activation of target organs. We plan 
to investigate the relationship between CNS NET-availability and peripheral NET (measured 
by whole-body ([11C]MRB PET imaging) and SNS activity (measured by MSNA) both in normal 
BMI and obese individuals. We propose that CNS NET-availability at rest in the fasting state 
in the hypothalamus and thalamus (brain regions involved with glucose and energy 
metabolism) will correlate with NET binding in BAT and skeletal muscle in individuals with 
normal BMI. In contrast, because of obesity-associated regionalized SNS dysfunction, CNS- 
NET availability will not correlate with peripheral NET-binding or MSNA in individuals with 
obesity.
High carbohydrate meal ingestion is a strong stimulus for activation of the SNS. Food 
intake (in particular carbohydrate-rich meals) is a strong stimulus for activation of SNS in both 
animals 38 and humans 39-42. This has been demonstrated by plasma NE tracer kinetics 38,39 
and MSNA 39,41-43. In rodents, NE turnover in BAT, but not in the spleen or heart, increased in 
response to a meal 38. Regionalized SNS activation was also observed in humans: increased 
in skeletal muscle and kidneys, but no or minimal change in heart and liver 39. SNS play a 
major role in activating BAT after a meal 44, and a recent study using PET Imaging showed 
increased BAT activation after ingestion of a high carbohydrate-rich meal 45.
Food-stimulated SNS activation in obesity. Studies using MSNA have shown that obesity 
is associated with SNS hyperactivity in the fasting state 17,18. However, food-stimulated 
increments in MSNA have been shown to be blunted in individuals with obesity 15,27. Yeckel et 
al. 28 measured sympathetic activity
during exercise with the exercise recovery 
index – calculated by a ratio of heart rate 
normalized
for
oxygen
consumption 
(VO2). Sympathetic activity in response to 
exercise was compared with metabolic 
response in response to an OGTT and a 
high-carbohydrate meal challenge. The 
authors observed that insulin levels after 
the OGTT and test meal correlated with 
degree of sympathetic overactivity (Figure 
9). Increased body fat (measured by 
densitometry)
was
associated
with 
exercise-induced sympathetic overactivity. 
Therefore, based on the premise that 
obesity is associated with fasting SNS
Figure 9. Relationship between sympathetic activity 
(HR/V˙ O2 plateau) and insulin levels OGTT and 
high-carbohydrate meal
hyperactivation and diminished increment in SNS activation in response to a food challenge 
(high-carbohydrate mixed meal test), we plan to measure [11C]MRB-binding in response to a 
meal in individuals with obesity in comparison to a group of individuals with normal BMI. In 
addition, to confirm the degree of SNS dysfunction in obesity, MSNA will be measured under 
similar circumstances (before and after a high-carbohydrate meal test).
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In our pilot study, sex differences were observed in response to a cold stimulus, with men 
showing a significantly increased [11C]MRB-binding in response to cold 12. Therefore, we will 
also investigate whether sex differences may play a role in [11C]MRB-binding in response to a 
food stimulus. To build on our preliminary studies, we plan to quantify NET in peripheral tissues 
(adipose tissue (BAT and WAT) and skeletal muscle) and CNS at rest in fasting and post-
prandial state, in both men and women with normal BMI and with obesity.
2.2
Background/prevalence of research topic
Obesity affects more than a third of the population of the United States 46 and carries an 
increased risk of developing type 2 diabetes, hypertension, heart disease, stroke, and certain 
types of cancers 47. However, the exact mechanisms by which obesity contributes to these 
cardiometabolic disorders remains uncertain. Sympathetic nervous system (SNS) dysfunction 
has been described in individuals with obesity 15-18 and it has been proposed as one of the 
mechanisms involved in the development of obesity-related cardiometabolic complications 
20,21. It was initially proposed that obesity was associated with decreased SNS activity with a 
consequent decrease in energy expenditure and weight gain 48. However, these findings were 
not confirmed by other reports. Some studies showed no change or even increased SNS 
activity (measured by plasma norepinephrine levels) 49. Studies using microneurography, a 
direct measurement of SNS activation, showed that individuals with obesity may have 
sympathetic overactivity 17,18. SNS activity may vary by tissue 21,50. Specifically: SNS activation 
increases in kidneys and skeletal muscle and decreases or is unchanged in heart, and mostly 
unchanged in adrenal glands. Regionalized SNS hyperactivity in kidneys and skeletal muscle 
may lead to hemodynamic and metabolic changes in individuals with obesity. This results in 
an increased risk of developing hypertension and diabetes, and, in turn, cardiovascular 
morbidity and mortality. However, it is not exactly clear the interplay between obesity and 
regionalized SNS dysfunction. We propose that obesity is associated with dysfunctional 
coordination of SNS activation; and to prove that, we plan to measure synchronous SNS 
activity in various target organs in healthy individuals with normal body mass index (BMI) and 
with obesity.
SNS activation of target organs is mediated principally by the neurotransmitter 
norepinephrine (NE). Noradrenergic neurons release NE in the neuronal synapse of target 
organs and NE reuptake by pre-synaptic norepinephrine transporters (NET) are important for 
regulating NE levels in the synapse and neurotransmission 22. NET availability is thus 
considered a marker of neuronal activation 22. Our group has validated the use of [11C]MRB 
which has high specificity for NET. In a rodent model, this tracer properly identified NET-rich 
regions in the body, such as brown adipose tissue (BAT), white adipose tissue (WAT), skeletal 
muscle, liver, heart, and brain 24. In humans, [11C]MRB has been used to quantify unstimulated 
BAT in men and women 51, and NET availability in BAT from healthy women with obesity was 
found to be reduced in comparison to healthy women with normal body mass index (BMI) 2. In 
addition, preliminary unpublished data from our group showed that, in women, NET
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availability in skeletal muscle (deltoid) inversely correlated with BMI. [11C]MRB PET imaging 
allow us to properly identify NET-rich regions in both in humans and animals and may 
be used as a novel technique to quantify SNS activation of target organs. Furthermore, 
we will investigate whether NET activity (as measured by [11C]MRB PET imaging) 
correlates with muscle sympathetic nerve activity (MSNA), the gold-standard 
measurement for systemic SNS activation. Altered coordinated SNS multi-organ activation 
in the fasting and post-prandial state in obesity may play a role in the pathophysiology of 
obesity-associated cardiometabolic disorders.
There is also compelling evidence that SNS activity is not only altered at baseline in obesity 
but also in response to stimuli. Studies have found that obesity is associated with altered SNS 
responses to various stimuli including oral glucose, food, hyperinsulinemic-euglycemic clamp 
15,27,28,52. Food-stimulated increments in MSNA have also been shown to be blunted in 
individuals with obesity 15,27. Thus, we plan to simultaneously measure NET-binding in BAT, 
WAT, skeletal muscle, and brain in the fasted state and after a high carbohydrate meal 
challenge. Furthermore, we will evaluate the relationship between NET-rich regions in the 
brain involved in energy homeostasis (such as hypothalamus) and motor and sensory relay 
(thalamus) with peripheral NET-rich tissues, such as BAT and skeletal muscle
3 Rationale/Significance
3.1
Problem Statement
Sympathetic nervous system (SNS) dysfunction has been observed in obesity and may play 
a role in the pathophysiology of obesity-associated cardiometabolic disorders.
3.2
Purpose of Study/Potential Impact
This project will help further understand the impact of obesity in SNS activity and better 
understand the role of the SNS in the development of cardiometabolic disorders in patients 
with obesity.
3.2.1
Potential Risks
Risks Associated with Radiation:
Yale University Radioactive Investigational Drug Committee (Yale RIDC) will review the use 
of radiation in this research study, and no subjects will be scanned until RIDC approval is 
obtained. This research study involves exposure to radiation from [11C]MRB PET scanning. 
This radiation exposure is not necessary for medical care and is for research purposes only.
For each individual PET scan, subjects will receive up to ≤ 20 mCi of, [11C]MRB, plus 
transmission scans. This is equal to an effective dose of 0.428 rem per injection.
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Although each organ will receive a different dose, the maximum amount of radiation exposure 
subjects will receive from participating in up to two PET scans is equal to an effective dose 
0.856 rem, for a total of up to 40 mCi of [11C]MRB in 2 injections. This calculated value is used 
to relate the dose received by each organ to a single value.
Subjects will also receive radiation exposure from low dose whole-body CT scans on the mCT, 
subjects will receive up to 1250 mrem (1.250 rem) from up to 2 low dose head CT scans (0.625 
rem each).
The maximum amount of radiation an individual subject could receive in this study is from up 
to 2 injections of ≤ 20 mCi of [11C]MRB, plus CT scans. This is equal to an ED of 2.106 rem.
The amount of radiation subjects will receive in this study is below the dose guidelines 
established by the RIDC guidelines for research subjects. This guideline sets an effective dose 
limit of 5 rem per year.
Adverse effects of the radiopharmaceuticals in this study have not been reported. However, 
the possibility exists for a rare reaction to any of the substances or procedures to which a 
subject is exposed.
Physical examination, venipuncture/IV placement, EKG, lab data collection and analysis: 
Subjects will receive a comprehensive medical history assessment, physical examination, 
EKG, and lab testing to ensure good physical health status and eligibility. These are routine 
medical procedures and should add no risks other than those normally associated with these 
procedures. Study doctors and/or nurse practitioners will assess the EKG and screening 
laboratory blood work.If any abnormal findings occur, they will provide appropriate medical 
advice. Potential participants who are excluded from the study due to medical reasons, or 
those who are in immediate need for medical or psychiatric attention, will be referred to the 
appropriate facilities at Yale New Haven Hospital so they can receive the clinical care needed 
to address their condition. Subjects will be exposed to the risk of venipuncture/IV placement 
that is routinely done in the HRU at the YNHH. Risks associated include: local bruising, 
hematoma, or infection. If this occurs, appropriate treatment will be provided immediately. On 
extremely rare occasions, a blood clot or infection might occur, and if this occurs, subjects will 
be treated at the HRU, which is fully staffed with nursing specialists and medical personnel. 
These risks are mitigated by the use of research HRU nurses who have extensive experience 
in venipuncture and IV catheterization. Transient vasovagal symptoms, e.g., nausea, 
sweating, and lightheadedness may also occur during IV catheter placement. Careful efforts to 
maintain confidentiality have been effective in previous research, and only patients’ code 
numbers will be recorded on forms to protect confidentiality. Efforts to protect confidentiality 
are further detailed below.
Oral glucose tolerance test (OGTT): The OGTT is a commonly used outpatient test for 
diagnosis of type 2 diabetes. It is not associated with any specific adverse effects. 
Occasionally, some patients may experience mild nausea or GI discomfort following drinking 
the 75 gram glucose drink; however, this typically resolves after eating food.
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Microneurography: The risk of microneurography is a peroneal paresthesia for 2-10 days 
following the experiment. The incidence of paresthesia is reported to be less than 10%, in less 
than 1% of all subjects paresthesia can persist for up to two months. Paresthesia resolves 
spontaneously. Microneurography can occasionally be painful, but this is very unusual.
Phlebotomy: The total amount of blood drawn is ~160 ml (including screening, OGTT). People 
who are in good health are not usually affected by this kind of blood loss. However, to be safe, 
subjects will be warned against donating blood for at least 6 weeks before and after completing 
this study. Additionally, subjects are screened for anemia at the first visit and females with a 
hematocrit less than 35% and males with hematocrit less than 40% will be excluded.
DXA scanning: DXA scanning for body composition is performed routinely by our group. The 
DEXA measurement poses no major risk to the subject. The amount of radiation the subject 
will be exposed to is small and is one tenth of the amount of radiation for a chest X-ray.
Medical treatment for injury: The consent form will specify that medical therapy will be 
provided for injuries sustained as a consequence of participation in this research. This will be 
provided at the HRU located at the YNHH or Church Street Research Unit (CSRU).
Minimizing Risks:
General: All investigators and research team members have taken the Human Investigations 
Training Course either on-line or in person at the Yale School of Medicine. Trained medical 
staff at the HRU will perform all clinical research procedures and all subjects will have health 
screens reviewed by a physician or nurse practitioner prior to their participation. In the event 
of an injury, medical therapy will be offered with the cost incurred by the subject’s medical 
insurance carrier. Confidentiality of all study information is maintained by identifying subjects 
by code numbers, which are subsequently linked to their data files. No individuals, other than 
professionals directly involved in the study will be allowed to read data forms to ensure 
confidentiality. No subjects are identified by name in published data and only by code in data 
storage areas.
Informed consent: All subjects are consented by one of the study investigators or research 
staff, as part of determining their study eligibility. The purpose, nature, and potential risks and 
benefits of the study will be explained to each potential subject in detail during the screening 
interview. Prior to initiating the study, one of the study investigators or research staff will 
explain the planned study interventions, and each subject will be asked to read and sign a 
consent form approved by the Yale School of Medicine Human Investigation Committee (HIC). 
All potential subjects are given ample time to ask questions and only after this are they asked 
to provide informed consent in the research study. Subjects are given a copy of the signed 
consent for their personal records.
IV Insertion and Blood Drawing: Risks of bruising, clotting, and infection will be minimized by 
having venipuncture performed by trained, experienced personnel under sterile conditions. To 
avoid injury due to fainting, the antecubital vein catheter will be inserted when the subjects are 
recumbent. Females with a hematocrit less than 35% and males with a hematocrit less than 
40% will be excluded. All subjects having donated blood within 6 weeks of the study will be
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asked to postpone study participation (with a repeat blood count prior to future enrollment), 
and patients will be advised to refrain from blood donation for 6 weeks after each study.
Radiation. The Yale-New Haven Hospital Radiation Safety Committee (RSC) will review the 
use of radiation in this research study, and no subjects will be enrolled until RSC approval is 
obtained. This research study involves exposure to radiation from DXA scan and [11C]MRB 
PET scanning. This radiation exposure is not necessary for medical care and is for research 
purposes only. The targeted amount of radiation an individual subject will receive in this study 
is from is from 2 injection(s) of ≤ 20 mCi of [11C]MRB plus transmission scans and CT scans 
CT scans for attenuation correction and DXA scan. All scans will be done in the presence of 
medical supervision and trained nursing staff in an institution specifically designed to support 
imaging studies. In the event of serious medical complications, the Yale University PET scan 
facilities have immediate access to or consultation with specialized medical units at the Yale- 
New Haven Hospital. Preparation of radiopharmaceuticals and performance of PET scans will 
be by radiochemists and technologists of the Department of Diagnostic Radiology, Yale 
University School of Medicine. These professionals are qualified by training and experience in 
the safe use and handling of radiopharmaceuticals. Subjects will be asked about their previous 
radiation exposure, and those who have had research exposure within the past year will be 
excluded if their cumulative annual exposure (including the present study) exceeds FDA limits. 
No PET studies will be performed on pregnant or potentially pregnant women, as confirmed by 
pregnancy testing during evaluation and on each scan day before initiation of any scan 
procedures. If subjects are breastfeeding they will not be able to participate in this research 
study.
Microneurography. The likelihood of the occurrence of symptoms following microneurography 
will be minimized by the following certain procedures. First, few problems or symptoms are 
reported in laboratories whose microneurographers have received training from individuals 
with a documented record of success and safety. Dr. Stone or Dr. Stachenfeld will perform all 
microneurography studies. Dr. Stone is a highly qualified microneurographer in the laboratory 
with over two years’ experience, and Dr. Stachenfeld has performed microneurography in her 
laboratory for 10 years, and this will reduce the risk of occurrence of symptoms following the 
experiment. The time spent searching for a nerve fascicle within the peroneal nerve will be 
limited to < 60 min. No nerve site will be sampled more than once. The recording electrodes 
are inspected under a dissecting microscope to ensure electrode tip integrity (those electrodes 
found with damaged tips are not used) and each recording electrode will be used only once 
for intraneural recording. Placement of the electrodes and positioning in the common peroneal 
nerve are performed under sterile conditions. Metal microelectrodes are steam sterilized at 
Yale New Haven Hospital before use. The site of electrode placement is cleaned with alcohol 
and only investigators wearing sterile gloves handle the sterile microelectrodes. Following 
these simple guidelines, the risk of occurrence of symptoms following microneurography will 
be small. Microneurography studies are conducted at the Pierce Laboratory.
3.2.2
Potential Benefits
The proposed studies do not have direct, short-term subject benefits. Assessment of risks vs. 
benefits requires some consideration of the potential benefits to society from the studies
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proposed. Obesity affects more than a third of the population of the United States and carries 
an increased risk of cardiometabolic disorders. Given the considerable potential benefit to 
society, we believe the risk-benefit ratio for participation in the proposed studies is favorable.
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4 Study Objectives
4.1
Hypothesis
Our hypothesis is that coordinated multi-organ response (brown and white adipose tissue, 
muscle, is modulated by the SNS both in the fasting state and after a meal; however, this 
coordinated response becomes dysfunctional in obesity. Dysfunctional brain regulation of 
SNS activation to target organs may explain the increased risk of cardiometabolic disorders 
in obesity.
4.2
Primary Objective
The primary objective of this study is to determine whether sympathetic nervous system 
(SNS) activity in adipose tissue (WAT and BAT), muscle and brain is altered in individuals 
with obesity in comparison to individuals with normal weight. Simultaneous multi-organ SNS 
activation will be obtained with a radiotracer for norepinephrine transporters (NET) for whole- 
body Positron Emission Tomography (PET) imaging and microneurography (gold standard 
test for assessment of muscle SNS activity).
4.3
Secondary Objectives (if applicable)
The secondary objectives of this study are: 1) to evaluate whether gender differences affect 
peripheral SNS in healthy normal weight and obese men and women in adipose tissue (WAT 
and BAT) and muscle (resting/fasting); 2) To investigate the relationship between peripheral 
and central SNS activity in obesity, by correlating SNS activity in peripheral tissues (WAT, 
BAT, and muscle) and brain; and 3) To investigate NET CNS and peripheral SNS activity 
before and after a high carbohydrate mixed meal in lean and obese men and women.
4.4
Exploratory Objectives (if applicable)
N/A
5 Study Design
5.1
General Design Description
Aim: To measure peripheral SNS in healthy normal weight and obese men and women in 
adipose tissue (WAT and BAT), deltoid skeletal muscle, and brain NET at fast and after a 
mixed meal test (MMT).
The goal of this study is to measure in 20 individuals with normal weight (BMI 18-25 kg/m2) 
and 20 individuals with obesity (BMI 30-50 kg/m2) NET-binding in WAT and BAT to better 
understand the role of SNS in obesity. SNS activation of a) peripheral tissues: adipose tissue 
(BAT and WAT) and skeletal muscle; and b) CNS NET will be measure synchronously by 
using [11C]MRB whole body
PET imaging in combination with the gold standard measurement of peripheral SNS activity 
(muscle sympathetic nervous activity (MSNA)). PET and MSNA visits do not need to occur in 
sequence, i.e. PET scans can occur before or after the MSNA.
Visit 1 - Screening procedures:
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Potential subjects will complete pre-screening over the telephone for approximately 10 minutes 
to determine preliminary eligibility based on inclusion/exclusion criteria and basic socio-
demographic information will be collected. Potentially qualifying participants will then be 
screened in person at the Yale New Haven Hospital (YNHH) Research Unit (HRU) or Church 
Street Research Unit (CSRU) with vital signs, EKG, and a medical history and physical exam. 
A urine pregnancy test will be administered for females.
Oral glucose tolerance test (OGTT) will be administered as follows: Following a 10-hour 
overnight fast, a nurse will insert an intravenous (IV) catheter. Subsequently, subjects will 
ingest 7.5 oz of Glucola, which contains 75 g of dextrose in orange flavored water. Blood 
samples will be taken at -15, 0, 10, 20, 30, 60, 90 and 120 minutes (after glucola ingestion) 
for the measurement of plasma glucose, insulin, and C-peptide concentrations, and 
calculation of measurements of insulin sensitivity index (Matsuda Index). Fasting blood 
obtained from the catheter will be collected for HbA1C, creatinine, liver enzymes (ALT, AST), 
TSH, hematocrit, and lipid panel. Approximately 40 mL of blood will be drawn at this visit.
Body Composition, Percent Body Fat & Percent Body Water: will be assessed using 
bioelectrical impedance analysis Tanita® scale (FDA cleared); which is a special multi 
frequency seg-mental body composition analyzer that delivers a very mild electrical current 
through the feet that allows measurement of fat mass, percent body fat, fat free mass, total 
body water, and percent body water. This test will be performed at screening visit. For better 
accuracy, measurement of percent fat and percent body water will be obtained with whole 
body Dual X-Ray Absorptiometry (DXA) scan (Hologic®) (located in the HRU) on screening- 
OGTT visit. The scanner arm will move over the participant’s body from feet to head. The 
machine uses a small amount of radiation (one tenth of the amount of radiation from a chest 
x-ray) to measure body fat, muscle and bone density.
Subjects who fail any of the screening procedures will be informed and excluded from 
participating in the study, however they will be allowed to be re-screened at a later 
time,
Visit 2 – Whole body PET Imaging Visit - FASTING:
Two weeks after the screening visit, participants who qualified for the study will be asked to 
come to the PET Center to undergo the whole body [11C]MRB PET scan. Participants will be 
asked to maintain their regular exercise routine.
Human PET-CT Protocol: [11C]MRB will be synthesized at the Yale PET center based on 
procedures described and previously performed 1,2. An intravenous bolus dose of MRB 
(~120mCi) will be injected by an infusion pump. PET data will be acquired dynamically for 120 
min using the Siemens mCT-X whole-body PET/CT scanner using continuous bed motion to 
image perform multiple PET bed acquisitions from the top of the head to the lower abdominal 
region. A CT scan will be performed for attenuation correction and to help delineate the BAT and 
other regions of interest (ROIs). Images will be reconstructed with an ordered subset 
expectation maximization algorithm using point spread function correction and time-of- flight 
information. To delineate fat (WAT, BAT) and skeletal muscle regions of interests (ROIs),
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CT images are first resliced to match the resolution and location of PET images. As performed 
in previous studies 2, a supraclavicular ROI is first manually drawn on the CT images. Within 
that area, the fat ROI is segmented using the CT images and an intensity window from -200 
to -50 HU. Subsequently the fat ROI is segmented into WAT and BAT ROIs by using the 11C- 
MRB PET images: Standardized Uptake Value (SUV) ≥ 1.25 for BAT, SUV < 1.25 for WAT.
Brain ROIs for predetermined NET-rich areas of the brain, including the locus ceruleus, raphe, 
thalamus and hypothalamus were defined in template space and are applied to the PET 
images by using the participant’s MR image as an intermediate step. For analysis of tracer 
uptake, mean SUV will be reported for segmentation of adipose tissue. Because the 
distribution volume ratio (DVR) is a more precise measurement of uptake for reversible tracers 
3 such as 11C-MRB, regional quantification of 11C-MRB uptake as DVR (VT/ VND), estimated via 
multilinear reference tissue model 2 (MRTM2) 4,5 (t*=30 min) using the occipital cortex, a region 
with low NET density 2,5,6 as the reference region. MRTM2 reduces the variability of MRTM (4) 
parameter estimates by fixing one of the three parameters, the parameter k2', which is only 
related to the reference region time-activity curve, to a common value for all target regions. In 
this study, the parameter k2' was fixed to 0.021 min-1, based on previous brain studies and 
previous BAT PET studies 2. In addition to DVR, MRTM2 can estimate R1, the relative 
radioligand delivery, using a 1 tissue compartment model (1TC). For the 1TC model, volume 
of distribution, VT = K1/k2, where K1 (mL/min−1/mL−1) is the rate constant of radiotracer from 
plasma to tissue of target organ and k2 is the rate constant from tissue to plasma. Using a 
reference region with non-displaceable binding (e.g. occipital lobe) we can estimate similar 
rate constants: VND = K’1/k’2. Recalling that K1 and K’1 are the rate constants for transfer from 
plasma to the tissue and reference region, respectively, then we can estimate relative 
radioligand delivery as R1 = K1/K’1.
The maximum number of attempts for each failed/incomplete imaging and test is 2 times.
Visit 3 – Whole body PET Imaging Visit - High-Carbohydrate Mixed Meal Test (MMT)
High Carbohydrate Mixed meal test (MMT): Two weeks after visit 2, participants will come to 
the PET Center after a 10-hour overnight fast. A nurse will insert an intravenous (IV) catheter 
and subjects will ingest a liquid meal (65% CHO, 20% fat, and 15% protein, equal to 40% of 
daily energy expenditure, per ~16 ounces) to be prepared by the Metabolic Kitchen located at 
the HRU. Blood samples will be collected at −10, 0, 15, 30, 60, 90, and 120 min) for 
measurement of glucose, insulin and C-peptide concentrations 7. Plasma samples will be 
drawn at baseline and throughout the MMT for measurement of additional hormones (such as 
leptin, ghrelin, thyroid hormones, and catecholamines) and free fatty acids (FFA) levels. Insulin 
and glucose will be used to calculate indexes of insulin sensitivity 7-9. Hormones, glucose and 
FFA will be correlated with PET measurements of brown fat at baseline and during the MMT. 
Approximately 60 mL of blood will be drawn at this visit.
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Participants will undergo whole body [11C]MRB PET scan as described in visit 3. After the 
MMT, PET data will be acquired dynamically for 120 min using the Siemens mCT-X whole- 
body PET/CT scanner after an intravenous bolus dose of MRB (~20mCi).
The order of the PET scan visits 2 and 3 will be randomized.
PET and MSNA visits do not need to occur in sequence, i.e. PET scans can occur before or 
after the MSNA.
Measurement of Energy Intake: Hunger and fullness will be determined immediately before, during 
and after the PET scans with Visual Analog Scales (VAS).
Visit 4 – Sympathetic Nervous System Activity (SNSA) measurement Visit - Fasting
Two weeks after visit 3, participants will be asked to return for a separated visit for 
measurements of SNS activity at rest and fasting. This visit will be performed at the Pierce 
Laboratory (Dr. Stachenfeld’s laboratory). The participants will come in the morning (~9am) 
and will be asked to fast for 10-hour and to maintain their regular exercise routine. The 
following measurements will be obtained: MSNA, heart rate (HR), blood pressure (BP), 
electrocardiogram (EKG), and heart rate variability. The proposed procedures will be 
performed at fasting (baseline). Participants will be asked to maintain their regular exercise 
routine.
Muscle sympathetic nerve activity (MSNA): Microneurography is the gold standard, most 
sensitive and reliable MSNA assessment in humans. The primary measure of sympathetic 
nervous system activity is peripheral nerve activity, as measured by MSNA.
Muscle sympathetic nerve activity will be quantified by identification and measurement of 
sympathetic bursts in the integrated neurogram and expressed as burst frequency (number of 
bursts per unit time). ECG surface electrodes are applied to the chest, an elastic band 
incorporating a strain gauge element is strapped around the thorax for recording respiratory 
movements, and a sensor cuff of a servopulse-plethysmography arterial pressure monitor 
(FINAPRES, Ohmeda) is placed on the middle phalanx of the long finger (held at heart level). 
The experiments are carried out in an environmentally controlled chamber at an ambient 
temperature of 27.0 ± 0.1 °C. The experiment will begin with a 60-minute control period. During 
such time the subjects rest in the reclined position.
Measurements of HR, BP, and EKG (beat-to-beat) will also be obtained during the procedure. 
An intravenous catheter will be inserted into a forearm vein for blood sampling. Following the 
instrumentation period, resting HR, pulse interval (PI), BP, and MSNA will be recorded for 10 
minutes. Following these measurements, plasma will be taken for measurements of insulin, 
glucose, free fatty acids, norepinephrine, and epinephrine levels. Approximately 20 mL of 
blood will be drawn at this visit. After the first blood sample and the 10-min rest phase, subjects 
will begin a seven-minute period of rest with paced breathing (12 breaths per minute) while 
HR, BP, PI and MSNA are measured. The subjects will synchronize breathing to a metronome 
set to a cadence of ~12 breaths per minute (0.2 Hz).
Procedure: Microneurography Recording of multiunit postganglionic muscle sympathetic 
nerve activity (MSNA) will be made from the common peroneal nerve as it winds around the 
fibular head with the subject in the supine position with the subject’s thigh comfortably
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supported. This procedure requires us to first trace the course of the nerve using small 
electrical stimuli applied to the surface of the skin over the nerve or use the ultrasound to 
visually assist. This allows the microneurographer to find and focus the recording electrode tip 
on a nerve fascicle associated with muscle sympathetic activity. The time required to focus 
the electrode on the nerve averages about 30 min and will not exceed 60 min. In optimal 
conditions the track of the nerve from superficial stimulation is accurate and the nerve remains 
in position during insertion. An uninsulated subcutaneous electrode will serve as the 
reference. Mechanoreceptor afferent activity can be recorded during light tapping on the 
muscle belly or passive muscle stretch. A suitable intrafascicular recording site for MSNA 
consists of regular bursts and synchronous with the cardiac cycle. The neural activity is 
amplified (2x104), filtered (0.5-5.0 kHz), rectified, and integrated. The amplified and filtered 
nerve signal is also led to an audio monitor and through a resistance capacitance circuit (time 
constant 100 ms). The integrated nerve signal is digitized at 400 Hz along with the ECG signal 
while respiratory and arterial pressure signals are also sampled at 400 Hz (MacLab 8s). Data 
for analysis is selected from periods of quiet breathing in which subjects were relaxed and not 
talking. Microneurography was approved under HIC protocols # 8079, #0512000875, 
#1609018353 and is currently used under # 2000020950.
Cardiovascular variables. Recorded with a 16-channel computerized data-acquisition system 
at a sampling Speed of 400 Hz (ADI Instruments PowerLab 9, Castle Hill, Australia). Beat-to- 
beat DBP, SBP and pulse interval (PI) are determined using peak detection algorithms (44). 
Brachial artery BP and HR are measured with an automated sphygmomanometer (Colin 
Medical Instruments Corp, Komaki, Japan). Heart rate and pulse interval will be determined 
beat-to-beat from lead II of the EKG recording.
Visit 5 – Sympathetic Nervous System Activity (SNSA) measurement Visit – Mixed Meal 
Test (MMT)
Two weeks after visit 4, participants will be asked to return for a separated visit for 
measurements of SNS activity at rest and after intake of liquid meal. Participants will undergo 
similar procedures described on visit 4. However, MSNA will be obtained after MMT. The 
participants will come in the morning (~9am) and will be asked to fast for 10-hour and to 
maintain their regular exercise routine. A nurse will insert an intravenous (IV) catheter and 
subjects will ingest a liquid meal (65% CHO, 20% fat, and 15% protein, equal to 40% of daily 
energy expenditure, per ~16 ounces) to be prepared by the Metabolic Kitchen. Measurements 
of HR, BP, and EKG (beat-to-beat) will be obtained during the procedure. An intravenous 
catheter will be inserted into a forearm vein for blood sampling. Following the instrumentation 
period, resting HR, pulse interval (PI), BP, and MSNA will be recorded for 10 minutes. 
Following these measurements, plasma will be taken for measurements of insulin, glucose, 
free fatty acids, norepinephrine, and epinephrine levels. Approximately 40 mL of blood will be 
drawn at this visit.
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Participants will be asked to maintain their regular exercise routine. The order of SNSA Fasting 
and MMT visits (visits 4 and 5) will be randomized. All study visits will be conducted within an 
8-week period (including the two PET and SNSA scans).
Schematic figure of the visits and procedures to be done. The order of the PET scans will be 
randomized.
Abbreviations: BP=blood pressure, EKG=electrocardiogram, HR=heart rate, MSNA=Muscle sympathetic nerve 
activity, SNSA=Sympathetic Nervous System Activity
Method of Assignment/Randomization- The order of the PET scan visits 2 (PET-CT 
Fasting) and 3 (PET/CT-MMT) will be randomly assigned (with a 50% chance of being 
assigned to each visit). SNSA visits 4 (fasting) and 5 (MMT) will be also be also be 
randomized (with a 50% chance of being assigned to each visit).
PET and MSNA visits do not need to occur in sequence, i.e. PET scans can occur 
before or after the MSNA.
5.1.1
Study Date Range and Duration
07/01/2021-06/30/2025
5.1.2
Number of Study Sites
1
5.2
Outcome Variables
Group differences in 1) [11C]MRB-binding in brown adipose tissue (BAT), white adipose tissue 
(WAT), skeletal muscle, and norepinephrine-rich brain regions (hypothalamus and thalamus);
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and 2) microneurography (muscle sympathetic nerve activity (MSNA)). Correlations between 
NET in the brain and peripheral NET and MSNA.
5.2.1
Primary Outcome Variables
The primary objective of this study is to determine whether sympathetic nervous system 
(SNS) activity in adipose tissue (WAT and BAT), muscle and brain is altered in individuals 
with obesity in comparison to individuals with normal weight. Simultaneous multi-organ SNS 
activation will be obtained with a radiotracer for norepinephrine transporters (NET) for whole- 
body Positron Emission Tomography (PET) imaging and microneurography (gold standard 
test for assessment of muscle SNS activity).
Primary outcome: SNS activity measured by [11C]MRB binding in adipose tissue, muscle and 
brain; and sympathetic burst in muscle (measured with microneurography).
5.2.2
Secondary Outcome Variables (if applicable)
The secondary objectives of this study are: 1) to evaluate whether gender differences affect 
peripheral SNS in healthy normal weight and obese men and women in adipose tissue (WAT 
and BAT) and muscle (resting/fasting); 2) To investigate the relationship between peripheral 
and central SNS activity in obesity, by correlating SNS activity in peripheral tissues (WAT, 
BAT, and muscle) and brain; and 3) To investigate NET CNS and peripheral SNS activity 
before and after a high carbohydrate mixed meal in lean and obese men and women.
5.2.3
Exploratory Outcome Variables (if applicable)
N/A
5.3
Study Population
The subjects we plan to enroll are in good medical health as evidenced by the medical 
history review during screening and lean (BMI 18.5-24.9 kg/m2) or non-diabetic obese (BMI 
30-50 kg/m2).
5.3.1
Number of Participants
We plan to enroll 20 participants with normal BMI (10 women and 10 men) and 20 with 
obesity (10 women and 10 men).
5.3.2
Eligibility Criteria/Vulnerable Populations 
Inclusion Criteria:
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In order to be eligible to participate in this study, an individual must meet all of the following 
criteria:
1.
Provision of signed and dated informed consent form
2.
Stated willingness to comply with all study procedures and availability for the duration 
of the study
3.
Age 18–45 years
4.
In good general health as evidenced by medical history and lean (BMI 18.5-24.9 kg/m2) 
or non-diabetic obese (BMI 30-50 kg/m2) with a fasting plasma glucose (FPG) <100 
mg/dL and a hemoglobin A1c <5.7%.
Exclusion Criteria:
An individual who meets any of the following criteria will be excluded from 
participation in this study:
1.
Hypertension
2.
Cardiac or pulmonary disease
3.
Known history of Type 1 or Type 2 diabetes
4.
Hepatic disease, swallowing and gastrointestinal disorders, including but not limited to 
diverticulitis and inflammatory bowel disease
5.
Disorders or impairment of the gag reflux
6.
Subjects with previous gastrointestinal surgery.
7.
Subjects with a pacemaker or other implanted electro medical device
8.
Current use of anti-obesity medications, supplements and/or anti-hyperglycemic 
medications
9.
Neurological injury or illness, and psychiatric medications
10. Women who are pregnant or lactating
11. Subjects who suffer from claustrophobia
12. Subjects who have received a diagnostic or therapeutic radiopharmaceutical within 7 
days prior to participation in this study
13. Subjects who work with radiation or have participated in other research studies 
involving ionizing radiation within one year of the PET scans that would cause the 
subject to exceed the yearly dose limits.
14. Subjects with history of IV drug use which would prevent venous access for PET tracer 
injection
15. Severe motor problems that prevent the subject from lying still for PET and MR imaging
16. Subjects who complain of chronic pain
17. Blood donation within 8 weeks of the study
18. Non-English speaking subjects
19. Subjects with poor venous access
20. Subjects with an allergy or intolerance to the carbohydrate meal or IV contrast.
21. Subjects with a history of bleeding disorders
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22. Subjects on anticoagulation medications.
23. Subjects who are Transgender
6 Methods
6.1
Treatment
6.1.1
Identity of Investigational Product
[11C]MRB will be prepared at the Yale PET Center under the supervision of Drs. Henry 
Huang and Nabeel Nabulsi in accordance with local Chemistry Manufacturing & Control 
(CMC) procedures and quality specifications described in our local Drug Master File (DMF), 
which has been approved by the Yale University Radioactive Drug Research Committee (YU 
RDRC).
6.1.2
Dosage, Administration, Schedule
N/A
6.1.3
Method of Assignment/Randomization
Method of Assignment/Randomization- The order of the PET scan visits 2 (PET-CT Fasting) 
and 3 (PET/CT-MMT) will be randomly assigned (with a 50% chance of being assigned to 
each visit). SNSA visits 4 (fasting) and 5 (MMT) will be also be also be randomized (with a 
50% chance of being assigned to each visit).
Blinding and Procedures for Unblinding 
N/A
6.1.4
Packaging/Labelling
N/A
6.1.5
Storage Conditions
N/A
6.1.6
Concomitant therapy
There are no restrictions.
6.1.7
Restrictions
There are no restrictions.
6.2
Assessments
Assessments are detailed in the figure below:
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Abbreviations: BP=blood pressure, EKG=electrocardiogram, HR=heart rate, MSNA=Muscle 
sympathetic nerve activity, SNSA=Sympathetic Nervous System Activity
Please note: The order of visits 2 and 3 are will be randomly assigned with a 50% chance of being 
assigned to each visit
6.2.1
Efficacy
See 5.1 General Design Description
6.2.2
Safety and Pregnancy-related policy
Risks Associated with Radiation:
No PET studies will be performed on pregnant or potentially pregnant women, as confirmed 
by pregnancy testing during evaluation and on each scan day before initiation of any scan 
procedures. If subjects are breastfeeding, they will not be able to participate in this research 
study.
6.2.3
Adverse Events Definition and Reporting
1.
Personnel responsible for the safety review and its frequency:
The principal investigator, Dr. Belfort De Aguiar will be responsible for monitoring the data, 
assuring protocol compliance, and conducting the safety reviews at the specified frequency, 
which must be conducted at a minimum of every 6 months (including when reapproval of the 
protocol is sought). During the review process, the principal investigator will evaluate 
whether the study should continue unchanged, require modification/amendment, or close to 
enrollment. Either the principal investigator, the HIC, or the RSC have the authority to stop or 
suspend the study or require modifications.
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2. The risks associated with the current study are deemed greater than minimal for the 
following reasons: (choose those that apply)
The total amount of radiation in this study is from up to 3 low dose CT scans - used to optimize 
the PET scan – and up to 3 injections of radioactive material (20 mCi), [11C]MRB and from a 
low dose CT scans used to help obtain the PET images and the DXA scan .
The average yearly background radiation in the United States is about 0.3300 mrem. The 
amount of additional radiation from participating in this study is about 2.307 rem. This is equal 
to about 7.69 years worth of natural radiation.
The amount of radiation subjects will receive in this study is below the dose guidelines 
established by the RIDC guidelines for research purposes. This guideline sets an effective 
dose limit of 5 rem per year.
Adverse effects of the radiopharmaceuticals in this study have not been reported. However, 
the possibility exists for a rare reaction to any of the substances or procedures to which a 
subject is exposed.
3. Attribution of Adverse Events:
Adverse events will be monitored for each subject participating in the study and attributed to 
the study procedures / design by the principal investigator Dr. Belfort De Aguiar, according to 
the following categories:
a.) Definite: Adverse event is clearly related to investigational procedures(s)/agent(s). 
b.) Probable: Adverse event is likely related to investigational procedures(s)/agent(s). 
c.) Possible: Adverse event may be related to investigational procedures(s)/agent(s).
d.) Unlikely: Adverse event is likely not to be related to the investigational 
procedures(s)/agent(s).
e.) Unrelated: Adverse event is clearly not related to investigational procedures(s)/agent(s).
4. Plan for Grading Adverse Events:
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The following scale will be used in grading the severity of adverse events noted during the 
study:
1.
Mild adverse event
2.
Moderate adverse event
3.
Severe
Plan for Determining Seriousness of Adverse Events:
Serious Adverse Events:
In addition to grading the adverse event, the PI will determine whether the adverse event 
meets the criteria for a Serious Adverse Event (SAE). An adverse event is considered 
serious if it results in any of the following outcomes:
1.
Death.
2.
A life-threatening experience in-patient hospitalization or prolongation of existing 
hospitalization.
3.
A persistent or significant disability or incapacity.
4.
A congenital anomaly or birth defect; OR
5.
Any other adverse event that, based upon appropriate medical judgment, may 
jeopardize the subject’s health and may require medical or surgical intervention to prevent 
one of the other outcomes listed in this definition.
An adverse event may be graded as severe but still not meet the criteria for a Serious 
Adverse Event. Similarly, an adverse event may be graded as moderate but still meet the 
criteria for an SAE. It is important for the PI to consider the grade of the event as well as its 
“seriousness” when determining whether reporting to the IRB is necessary.
6. Plan for reporting UPIRSOs (including Adverse Events) to the IRB
The principal investigator will report the following types of events to the IRB: 
Any incident, experience or outcome that meets ALL 3 of the following criteria:
1.
Is unexpected (in terms of nature, specificity, severity, or frequency) given (a) the 
research procedures described in the protocol-related documents, such as the IRB-approved 
protocol and informed consent document and (b) the characteristics of the subject
population being studied; AND
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2.
Is related or possibly related to participation in the research (possibly related means 
there is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research); AND
3.
Suggests that the research places subjects or others at greater risk of harm 
(including physical, psychological, economic, legal, or social harm) than was previously 
known or recognized.
Unanticipated Problems Involving Risks to Subjects or Others (UPIRSOs) may be medical or 
non-medical in nature, and include – but are not limited to – serious, unexpected, and 
related adverse events and unanticipated adverse device effects. Please note that adverse 
events are reportable to the IRB as UPIRSOs only if they meet all 3 criteria listed above.
These UPIRSOs/SAEs will be reported to the IRB in accordance with IRB Policy 710, using 
the appropriate forms found on the website. All related events involving risk but not meeting 
the prompt reporting requirements described in IRB Policy 710 should be reported to the IRB 
in summary form at the time of continuing review. If appropriate, such summary may be a 
simple brief statement that events have occurred at the expected frequency and level of 
severity as previously documented. In lieu of a summary of external events, a current DSMB 
report can be submitted for research studies that are subject to oversight by a DSMB (or 
other monitoring entity that is monitoring the study on behalf of an industry sponsor).
7. Plan for reporting adverse events to co-investigators on the study, as appropriate the 
protocol’s research monitor(s), e.g., industrial sponsor, Yale Cancer Center Data and Safety 
Monitoring Committee (DSMC), Protocol Review Committee (PRC), DSMBs, study 
sponsors, funding and regulatory agencies, and regulatory and decision-making bodies.
For the current study, the following individuals, funding, and/or regulatory agencies will be 
notified:

All Co-Investigators listed on the protocol.

National Institute of Health
1 Other Data Safety Monitoring Board (DSMB) or Committee (DSMC): The Yale PET 
Center’s internal Data Safety Monitoring Board, composed of the Directors of the PET 
Center, also meets to review any adverse events on a monthly basis.
1 Yale New-Haven Radiation Safety Committee (if applicable) 
Yale University Radiation Safety Committee (if applicable)
The principal investigator Dr. Belfort De Aguiar will conduct a review of all adverse events 
upon completion of every study subject. The principal investigator will evaluate the frequency
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and severity of the adverse events and determine if modifications to the protocol or consent 
form are required.
Research staff will call subject 1-3 days after the PET scan to check in and address any 
adverse events that may have arisen post PET scan.
The study will be stopped if either of the following occur:
1) If 1 or more subjects experiences a serious adverse event (SAE), which is considered by 
the investigator to be possibly or probably related to the radiotracer.
2) If 2 or more subjects experience AEs of severe intensity, which are considered by the 
investigator to be possibly or probably related to the radiotracer and of clinical concern.
After review of the safety data, SAEs and AEs confirmed to have been related to the 
radiotracer will be reported to the FDA and the appropriate regulatory committees at Yale. 
The study will be placed on hold until a determination is made as to whether the study may 
continue as written, if modifications are needed, or if the study will be discontinued.
Definitions
Adverse event (AE) means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 (a)).
An AE or suspected adverse reaction is considered "serious" (SAE) if, in the view of either 
the investigator or sponsor, it results in any of the following outcomes:

death,

a life-threatening adverse event,

inpatient hospitalization or prolongation of existing hospitalization,

a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions,

a congenital anomaly/birth defect, or

An important medical event that may not result in death, be life-threatening, or 
require hospitalization may be considered serious when, based upon appropriate 
medical judgment, they may jeopardize the participant and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. Examples 
of such medical events include allergic bronchospasm requiring intensive treatment 
in an emergency room or at home, blood dyscrasias or convulsions that do not result 
in inpatient hospitalization, or the development of drug dependency or drug abuse.
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Severity
Adverse events will be graded according to [name grading scale, e.g. CTCAE v5.0]. For AEs 
not included in the protocol defined grading system, the following guidelines will be used to 
describe severity.

Mild – Events require minimal or no treatment and do not interfere with the 
participant’s daily activities.

Moderate – Events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with 
functioning.

Severe – Events interrupt a participant’s usual daily activity and may require systemic 
drug therapy or other treatment. Severe events are usually potentially life-threatening 
or incapacitating. Of note, the term “severe” does not necessarily equate to 
“serious”.]
Relationship to Investigational Product
All AEs must have their relationship to study intervention assessed by the clinician who 
examines and evaluates the participant based on temporal relationship and his/her clinical 
judgment. The degree of certainty about causality will be graded using the categories below. 
In a clinical trial, the study product must always be suspect.

Definitely Related – There is clear evidence to suggest a causal relationship, and 
other possible contributing factors can be ruled out. The clinical event, including an 
abnormal laboratory test result, occurs in a plausible time relationship to study 
intervention administration and cannot be explained by concurrent disease or other 
drugs or chemicals. The response to withdrawal of the study intervention 
(dechallenge) should be clinically plausible. The event must be pharmacologically or 
phenomenologically definitive, with use of a satisfactory rechallenge procedure if 
necessary.

Probably Related – There is evidence to suggest a causal relationship, and the 
influence of other factors is unlikely. The clinical event, including an abnormal 
laboratory test result, occurs within a reasonable time after administration of the 
study intervention, is unlikely to be attributed to concurrent disease or other drugs or 
chemicals, and follows a clinically reasonable response on withdrawal (dechallenge). 
Rechallenge information is not required to fulfill this definition.

Potentially Related – There is some evidence to suggest a causal relationship (e.g., 
the event occurred within a reasonable time after administration of the trial 
medication). However, other factors may have contributed to the event (e.g., the 
participant’s clinical condition, other concomitant events). Although an AE may rate 
only as “possibly related” soon after discovery, it can be flagged as requiring more 
information and later be upgraded to “probably related” or “definitely related”, as 
appropriate.
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
Unlikely to be related – A clinical event, including an abnormal laboratory test result, 
whose temporal relationship to study intervention administration makes a causal 
relationship improbable (e.g., the event did not occur within a reasonable time after 
administration of the study intervention) and in which other drugs or chemicals or 
underlying disease provides plausible explanations (e.g., the participant’s clinical 
condition, other concomitant treatments).

Not Related – The AE is completely independent of study intervention administration, 
and/or evidence exists that the event is definitely related to another etiology. There 
must be an alternative, definitive etiology documented by the clinician.
Expectedness
The Principal Investigator will be responsible for determining whether an AE is expected or 
unexpected. An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the study 
intervention.
6.2.4
Pharmacokinetics (if applicable)
N/A
6.2.5
Biomarkers (if applicable)
N/A
6.3
Study Procedures
Visit 1 - Screening procedures:
Potential subjects will complete pre-screening over the telephone for approximately 10 
minutes to determine preliminary eligibility based on inclusion/exclusion criteria and basic 
socio-demographic information will be collected. Potentially qualifying participants will then 
be screened in person at the Yale New Haven Hospital (YNHH) Research Unit (HRU) with 
vital signs, EKG, and a medical history and physical exam. Fasting blood will be collected 
for HbA1C, creatinine, liver enzymes (ALT, AST), TSH, hematocrit, and lipid panel. A urine 
pregnancy test will be administered.
Oral glucose tolerance test (OGTT) will be administered as follows: Following a 10-hour 
overnight fast, a nurse will insert an intravenous (IV) catheter. Subsequently, subjects will 
ingest 7.5 oz of Glucola, which contains 75 g of dextrose in orange flavored water. Blood 
samples will be taken at -15, 0, 10, 20, 30, 60, 90 and 120 minutes (after glucola ingestion) 
for the measurement of plasma glucose, insulin, and C-peptide concentrations, and 
calculation of measurements of insulin sensitivity index (Matsuda Index). Approximately 40 
mL of blood will be drawn at this visit.
Body Composition, Percent Body Fat & Percent Body Water: will be assessed using 
bioelectrical impedance analysis Tanita® scale (FDA cleared); which is a special multi 
frequency seg-mental body composition analyzer that delivers a very mild electrical current
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that allows measurement of fat mass, percent body fat, fat free mass, total body water, and 
percent body water. This test will be performed at screening visit. For better accuracy, 
measurement of percent fat and percent body water will be obtained with whole body Dual
X-Ray Absorptiometry (DXA) scan (Hologic®) (located in the HRU) on screening-OGTT visit. 
The scanner arm will move over the participant’s body from feet to head. The machine uses 
a small amount of radiation (one tenth of the amount of radiation from a chest x-ray) to 
measure body fat, muscle and bone density.
Visit 2 – Whole body PET/CT Imaging Visit - FASTING:
After 2 weeks from visit 1, -participants who qualified for the study will be asked to come to 
the PET Center to undergo the whole body [11C]MRB PET scan. Participants will be asked 
to maintain their regular exercise routine.
Human PET-CT Protocol: [11C]MRB will be synthesized at the Yale PET center based on 
procedures described and previously performed 1,2. An intravenous bolus dose of MRB 
(~120mCi) will be injected by an infusion pump. PET data will be acquired dynamically for 
120 min using the Siemens mCT-X whole-body PET/CT scanner using continuous bed 
motion to image perform multiple PET bed acquisitions from the top of the head to the lower 
abdominal region. A CT scan will be performed for attenuation correction and to help 
delineate the BAT and other regions of interest (ROIs). Images will be reconstructed with an 
ordered subset expectation maximization algorithm using point spread function correction 
and time-of-flight information. To delineate fat (WAT, BAT) and skeletal muscle regions of 
interests (ROIs), CT images are first resliced to match the resolution and location of PET 
images. As performed in previous studies 2, a supraclavicular ROI is first manually drawn on 
the CT images. Within that area, the fat ROI is segmented using the CT images and an 
intensity window from -200 to -50 HU. Subsequently the fat ROI is segmented into WAT and 
BAT ROIs by using the 11C-MRB PET images: Standardized Uptake Value (SUV) ≥ 1.25 for 
BAT, SUV < 1.25 for WAT.
Brain ROIs for predetermined NET-rich areas of the brain, including the locus ceruleus, 
raphe, thalamus and hypothalamus were defined in template space and are applied to the 
PET images by using the participant’s MR image as an intermediate step. For analysis of 
tracer uptake, mean SUV will be reported for segmentation of adipose tissue. Because the 
distribution volume ratio (DVR) is a more precise measurement of uptake for reversible 
tracers 3 such as 11C-MRB, regional quantification of 11C-MRB uptake as DVR (VT/ VND), 
estimated via multilinear reference tissue model 2 (MRTM2) 4,5 (t*=30 min) using the 
occipital cortex, a region with low NET density 2,5,6 as the reference region. MRTM2 
reduces the variability of MRTM (4) parameter estimates by fixing one of the three 
parameters, the parameter k2', which is only related to the reference region time-activity 
curve, to a common value for all target regions. In this study, the parameter k2' was fixed to
0.021 min-1, based on previous brain studies and previous BAT PET studies 2. In addition to
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DVR, MRTM2 can estimate R1, the relative radioligand delivery, using a 1 tissue 
compartment model (1TC). For the 1TC model, volume of distribution, VT = K1/k2, where K1 
(mL/min−1/mL−1) is the rate constant of radiotracer from plasma to tissue of target organ 
and k2 is the rate constant from tissue to plasma. Using a reference region with non- 
displaceable binding (e.g. occipital lobe) we can estimate similar rate constants: VND = 
K’1/k’2. Recalling that K1 and K’1 are the rate constants for transfer from plasma to the 
tissue and reference region, respectively, then we can estimate relative radioligand delivery 
as R1 = K1/K’1.
Visit 3 – Whole body PET/CT Imaging Visit - High-Carbohydrate Mixed Meal Test (MMT) 
High Carbohydrate Mixed meal test (MMT):
After 2 weeks from visit 2, participants will come to the PET Center after a 10-hour overnight 
fast. A nurse will insert an intravenous (IV) catheter and subjects will ingest a liquid meal 
(65% CHO, 20% fat, and 15% protein, equal to 40% of daily energy expenditure, per ~16 
ounces) to be prepared by the Metabolic Kitchen located at the HRU. Blood samples will be 
collected at −10, 0, 15, 30, 60, 90, and 120 min) for measurement of glucose, insulin and C- 
peptide concentrations 7. Plasma samples will be drawn at baseline and throughout the 
MMT for measurement of additional hormones (such as leptin, ghrelin, thyroid hormones, 
and catecholamines) and free fatty acids (FFA) levels. Insulin and glucose will be used to 
calculate indexes of insulin sensitivity 7-9. Hormones, glucose and FFA will be correlated 
with PET measurements of brown fat at baseline and during the MMT. Approximately 60 mL 
of blood will be drawn at this visit.
Participants will undergo whole body [11C]MRB PET scan as described in visit 3. After the 
MMT, PET data will be acquired dynamically for 120 min using the Siemens mCT-X whole- 
body PET/CT scanner after an intravenous bolus dose of MRB (~20mCi).
The order of the PET scan visits 2 and 3 will be randomized.
Measurement of Energy Intake: Hunger and fullness will be determined immediately before, 
during and after the PET scans with Visual Analog Scales (VAS).
Visit 4 – Sympathetic Nervous System Activity (SNSA) measurement Visit - Fasting 
After 2 weeks from visit 3, participants will be asked to return for a separated visit for
measurements of SNS activity at rest and fasting. This visit will be performed at the Pierce 
Laboratory (Dr. Stachenfeld’s laboratory). The participants will come in the morning (~9am) 
and will be asked to fast for 10-hour and to maintain their regular exercise routine. The 
following measurements will be obtained: MSNA, heart rate (HR), blood pressure (BP), 
electrocardiogram (EKG), and heart rate variability,. The proposed procedures will be 
performed at fasting (baseline). Participants will be asked to maintain their regular exercise 
routine.
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Muscle sympathetic nerve activity (MSNA): Microneurography is the gold standard, most 
sensitive and reliable MSNA assessment in humans. The primary measure of sympathetic 
nervous system activity is peripheral nerve activity, as measured by MSNA.
Muscle sympathetic nerve activity will be quantified by identification and measurement of 
sympathetic bursts in the integrated neurogram and expressed as burst frequency (number 
of bursts per unit time). ECG surface electrodes are applied to the chest, an elastic band 
incorporating a strain gauge element is strapped around the thorax for recording respiratory 
movements, and a sensor cuff of a servopulse-plethysmography arterial pressure monitor 
(FINAPRES, Ohmeda) is placed on the middle phalanx of the long finger (held at heart 
level). The experiments are carried out in an environmentally controlled chamber at an 
ambient temperature of 27.0 ± 0.1 °C. The experiment will begin with a 60-minute control 
period. During such time the subjects rest in the reclined position.
Measurements of HR, BP, and EKG (beat-to-beat) will also be obtained during the 
procedure. An intravenous catheter will be inserted into a forearm vein for blood sampling. 
Following the instrumentation period, resting HR, pulse interval (PI), BP, and MSNA will be 
recorded for 10 minutes. Following these measurements, plasma will be taken for 
measurements of insulin, glucose, free fatty acids, norepinephrine, and epinephrine levels. 
Approximately 20 mL of blood will be drawn at this visit. After the first blood sample and the 
10-min rest phase, subjects will begin a seven-minute period of rest with paced breathing 
(12 breaths per minute) while HR, BP, PI and MSNA are measured. The subjects will 
synchronize breathing to a metronome set to a cadence of ~12 breaths per minute (0.2 Hz).
Procedure: Microneurography Recording of multiunit postganglionic muscle sympathetic 
nerve activity (MSNA) will be made from the common peroneal nerve as it winds around the 
fibular head with the subject in the supine position with the subject’s thigh comfortably 
supported. This procedure requires us to first trace the course of the nerve using small 
electrical stimuli applied to the surface of the skin over the nerve or use the ultrasound to 
visually assist. This allows the microneurographer to find and focus the recording electrode 
tip on a nerve fascicle associated with muscle sympathetic activity. The time required to 
focus the electrode on the nerve averages about 30 min and will not exceed 60 min. In 
optimal conditions the track of the nerve from superficial stimulation is accurate and the 
nerve remains in position during insertion. An uninsulated subcutaneous electrode will serve 
as the reference. Mechanoreceptor afferent activity can be recorded during light tapping on 
the muscle belly or passive muscle stretch. A suitable intrafascicular recording site for 
MSNA consists of regular bursts and synchronous with the cardiac cycle. The neural activity 
is amplified (2x104), filtered (0.5-5.0 kHz), rectified, and integrated. The amplified and 
filtered nerve signal is also led to an audio monitor and through a resistance capacitance 
circuit (time constant 100 ms). The integrated nerve signal is digitized at 400 Hz along with 
the ECG signal while respiratory and arterial pressure signals are also sampled at 400 Hz 
(MacLab 8s). Data for analysis is selected from periods of quiet breathing in which subjects 
were relaxed and not talking. Microneurography was approved under HIC protocols # 8079, 
#0512000875, #1609018353 and is currently used under # 2000020950.
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Cardiovascular variables. Recorded with a 16-channel computerized data-acquisition system 
at a sampling Speed of 400 Hz (ADI Instruments PowerLab 9, Castle Hill, Australia). Beat-to- 
beat DBP, SBP and pulse interval (PI) are determined using peak detection algorithms (44). 
Brachial artery BP and HR are measured with an automated sphygmomanometer (Colin 
Medical Instruments Corp, Komaki, Japan). Heart rate and pulse interval will be determined 
beat-to-beat from lead II of the EKG recording.
Visit 5 – Sympathetic Nervous System Activity (SNSA) measurement Visit – Mixed Meal 
Test (MMT)
After 2 weeks from visit 4, participants will be asked to return for a separated visit for 
measurements of SNS activity at rest and after intake of liquid meal. Participants will 
undergo similar procedures described on visit 4. However, MSNA will be obtained after 
MMT. The participants will come in the morning (~9am) and will be asked to fast for 10-hour 
and to maintain their regular exercise routine. A nurse will insert an intravenous (IV) catheter 
and subjects will ingest a liquid meal (65% CHO, 20% fat, and 15% protein, equal to 40% of 
daily energy expenditure, per ~16 ounces) to be prepared by the Metabolic Kitchen.
Measurements of HR, BP, and EKG (beat-to-beat) will be obtained during the procedure. An 
intravenous catheter will be inserted into a forearm vein for blood sampling. Following the 
instrumentation period, resting HR, pulse interval (PI), BP, and MSNA will be recorded for 10 
minutes. Following these measurements, plasma will be taken for measurements of insulin, 
glucose, free fatty acids, norepinephrine, and epinephrine levels. Approximately 40 mL of 
blood will be drawn at this visit.
Participants will be asked to maintain their regular exercise routine. The order of SNSA 
Fasting and MMT visits (visits 4 and 5) will be randomized. All study visits will be conducted 
within an 8-week period (including the two PET and SNSA scans).
6.3.1
Study Schedule
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Abbreviations: BP=blood pressure, EKG=electrocardiogram, HR=heart rate, MSNA=Muscle sympathetic nerve 
activity, SNSA=Sympathetic Nervous System Activity
6.3.2
Informed Consent
All subjects are consented by one of the study investigators or research staff, as part of 
determining their study eligibility. The purpose, nature, and potential risks and benefits of the 
study will be explained to each potential subject in detail during the screening interview.
Prior to initiating the study, one of the study investigators or research staff will explain the 
planned study interventions, and each subject will be asked to read and sign a consent form 
approved by the Yale School of Medicine Human Investigation Committee (HIC).
All potential subjects are given ample time to ask questions and only after this are they 
asked to provide informed consent in the research study. Subjects are given a copy of the 
signed consent for their personal records.
6.3.3
Screening
Research team members will contact interested potential participants to assess eligibility and 
provide the participant with additional information about the study, including the study 
procedures, purpose and potential complications. The brief screen via email or phone will 
ask for the following information to confirm study eligibility:

Name

Age/Date of Birth
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
Place of Birth

Phone Number/Email

Address

Marital Status

Race/Ethnicity

Height/Weight

Medical/Surgical History

Medications

Drug/Alcohol Use
In order to determine the potential participant’s eligibility and avoid unnecessary screening 
and traveling efforts, it is necessary to collect this information prior to having the subject 
arrive for an in-person screening visit. This information will be protected according to HIPAA 
policies and will be destroyed if the individual is determined to not be eligible for the study 
during this correspondence, or is otherwise not interested in participating in the study, unless 
the subject consents for his/her screening information to be stored for future studies that 
he/she may be eligible for.
During the in-person screening visit, the participant will arrive to the Yale New Haven 
Hospital (YNHH) Research Unit (HRU). The study nurse will collect vital signs, EKG, and the 
Principal Investigator will review the participant’s medical history and perform a physical 
exam. Fasting blood will be collected for HbA1C, creatinine, liver enzymes (ALT, AST), TSH, 
hematocrit, and lipid panel. A urine pregnancy test will be administered to females of child 
bearing potential.
Oral glucose tolerance test (OGTT) will be administered as follows: Following a 10-hour 
overnight fast, a nurse will insert an intravenous (IV) catheter. Subsequently, subjects will 
ingest 7.5 oz of Glucola, which contains 75 g of dextrose in orange flavored water. Blood 
samples will be taken at -15, 0, 10, 20, 30, 60, 90 and 120 minutes (after glucola ingestion) 
for the measurement of plasma glucose, insulin, and C-peptide concentrations, and 
calculation of measurements of insulin sensitivity index (Matsuda Index).
Body Composition, Percent Body Fat & Percent Body Water: will be assessed using 
bioelectrical impedance analysis Tanita® scale (FDA cleared); which is a special multi 
frequency seg-mental body composition analyzer that delivers a very mild electrical current 
that allows measurement of fat mass, percent body fat, fat free mass, total body water, and 
percent body water. This test will be performed at screening visit. For better accuracy, 
measurement of percent fat and percent body water will be obtained with whole body Dual
X-Ray Absorptiometry (DXA) scan (Hologic®) (located in the HRU) on screening-OGTT visit. 
The scanner arm will move over the participant’s body from feet to head. The machine uses 
a small amount of radiation (one tenth of the amount of radiation from a chest x-ray) to 
measure body fat, muscle and bone density.
6.3.4
Enrollment
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Research team members will enroll subjects who have been consented and screened during 
the in-person screening visit to ensure they meet eligibility criteria.
Recruitment Procedures:
Subjects will be recruited through flyers, by word of mouth, and through YCCI email blasts 
and JDAT.
a. Messaging to Study Participants through JDAT:
The following wording will be used for all applicable recruitment methods:
“You are receiving this [notification, email, letter, phone call] because you may qualify for 
and be interested in a research study conducted by Yale University investigators looking at 
whether obesity is associated with increased activity of the sympathetic nervous system.
For JDAT queries only: The Yale New Haven Health electronic health record system has 
searched medical conditions to find people who may be good matches for research studies. 
No one has looked at your record and no information has been shared with any research 
doctor or research team member. Just because you received this message does not mean 
that you are in a research study or that you have to decide to be in this or any study.
To opt-out of research, including opting out of receiving future messages about research 
studies, please email optout@yale.edu or call 1-877-978-8348 and select option #3.
Title of study: Sympathetic Activation in Obesity
Principal Investigator: Renata Belfort de Aguiar, MD, PhD 
Study Coordinator: Jacqueline Prinz
Phone # 203-785-5977
Description of Study:
The primary objective of this study is to determine whether sympathetic nervous system 
(SNS) activity in white adipose tissue (WAT) and brown adipose tissue (BAT), skeletal 
muscle and brain is altered in individuals with obesity in comparison to individuals with 
normal weight. Simultaneous multi-organ SNS activation will be obtained with a radiotracer 
for norepinephrine transporter (NET) for whole-body Positron Emission Tomography (PET) 
imaging and microneurography (peroneal muscle SNS activity).
Confidentiality and Privacy: Any personal health, financial data, and other information 
gathered in the study will remain confidential and will be stored on a password-protected 
computer, only accessed by study personnel. When the results of the research are published 
or discussed, no information will be included that would reveal your identity. We understand 
that information about you obtained in connection with your health is personal, and we are 
committed to protecting the privacy of that information. If you would like to learn more about 
participating in this study, please contact the study coordinator at 203-785-5977.
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Possible Benefits: The study may have no direct benefit to you, however general 
advancement of scientific knowledge from this study may include a better understanding of 
the importance of SNS activation in obesity.
Participation in this study is completely voluntary. You are free to decline to participate, to 
end participation at any time for any reason, or to refuse to answer any individual question at 
any time. Refusing to participate will involve no penalty or loss of benefits to which you are 
otherwise entitled (such as your health care outside the study, the payment for your health 
care, and your health care benefits).
Questions: If you have any further questions about this study, you may contact the 
investigator, Dr. Renata Belfort de Aguiar, at 203-737-4777. If you would like to talk with 
someone other than the researchers to discuss problems, concerns, and questions you may 
have concerning this research, or to discuss your rights as a research subject, you may 
contact the Yale Human Investigation Committee at (203) 785-4688.”
b.
Describe how potential subjects are contacted. The subjects will be asked to call us 
at our main recruitment number or email if they are interested in participating in the research 
study. Subjects will be contacted back for a confidential phone screening which leads to an 
in-person screening if eligible.
c.
Who is recruiting potential subjects? The PI, in collaboration with study investigators 
and trained research assistants, is responsible for subject recruitment.
6.3.5
On Study Visits
Screening

Consent

Urine Pregnancy test for women of child-bearing potential

Height and Weight using Tanita Scale

EKG

IV Insertion and OGTT

Blood draw for anemia, kidney and liver function and thyroid test and diabetes

Physical & Medical History

DXA Scan 
Visit 2 & 3 PET Scans

Subject will arrive to PET Center, fasted

Urine Pregnancy Test for women of child bearing potential

Urine Drug Screen

A-line and IV insertion

Mixed Meal Test on Visit 3

A low dose CT scan will be completed immediately before or after each PET 
scan.

Up to 3-4 hour PET scan

Collection of Blood Sampled during PET scan

Light Lunch given to subject after PET Scan 
Visits 4 & 5
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
Subject will arrive fasted to the Pierce Laboratory for measurements SNS activity

Urine Sample Collection

Collection of Weight

Collection of the following: Microneurography, BP, HR

EKG

IV Insertion

Blood Sample

Measurement of skin blood flow

Sympathetic Nervous System Activity (SNSA) measurement
Abbreviations: BP=blood pressure, EKG=electrocardiogram, HR=heart rate, MSNA=Muscle sympathetic nerve 
activity, SNSA=Sympathetic Nervous System Activity
6.3.6
End of Study and Follow-up
 Follow Up Procedures:
A member of the study team will reach out to the subject 1-2 weeks upon study completion 
for a brief follow-up telephone call. During this phone call, the subject will be asked how they 
are feeling since completing the study assessments and if they have any questions or 
concerns.
6.3.7
Removal of subjects
Participating in this study is voluntary. Subjects are free to choose not to take part in this 
study. Refusing to participate will involve no penalty or loss of benefits otherwise entitled to 
the subject (such as health care outside the study, the payment for health care, and health 
care benefits).
Subjects may withdraw permission to use and disclose health information at any time by 
telling the study staff or by writing to;
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Renata Belfort De Aguiar, M.D., Ph.D.
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Yale University School of Medicine 
300 Cedar street, TAC S-135
PO Box 208020
New Haven, CT 06520
The researchers may withdraw a subject’s participation in the research if deemed necessary. 
The subject may be asked to withdrawal from the study if they experience significant 
discomfort during the procedures, become pregnant during the study intervention, are not 
able to follow instructions during the procedures, or are not compliant with scheduled 
appointments.
No new health information identifying the subject will be gathered after the date of study 
withdrawal. Information that has already been gathered may still be used and given to others 
until the end of the research study, as necessary to ensure the integrity of the study and/or 
study oversight.
6.4
Statistical Method
6.4.1
Statistical Design
Primary endpoints: Group differences in 1) [11C]MRB-binding in brown adipose tissue (BAT), 
white adipose tissue (WAT), skeletal muscle, and norepinephrine-rich brain regions 
(hypothalamus and thalamus); and 2) microneurography (muscle sympathetic nerve activity 
(MSNA)). Correlations between NET in the brain and peripheral NET and MSNA.
Secondary endpoints: Plasma hormone levels, heart rate, blood pressure, electrocardiogram 
(beat-to-beat)..
Statistical Analysis: Before statistical testing, all continuous variables will be tested for 
normality of distribution. Summary statistics will be computed. For between-group comparisons 
of patient characteristics, two sample t-test or Mann-Whitney U test will be used for continuous 
variables, and Fisher’s exact test for categorical variables. Comparisons of repeatedly 
measured MSNA will be made using the mixed model analysis. Fixed factors include group 
and sex and their interactions. Linear contrasts comparing differences between groups or 
different time points within a group will be estimated.
6.4.2
Sample Size Considerations
Statistical Considerations: sample sizes of 6 per group (normal BMI x obesity, and women 
x men) will achieve 90% power to detect a between-group difference. To account for up to 
10% missing data due to drop out or poor-quality data, we will need to enroll a total of 40 
subjects (10 per group) for this study.
Fasting: A Power analysis was performed based on previous study published from our group 
in lean and obese female individuals: (lean 1.15 ±0.19 vs obese 0.80 ± 0.12 BAT DVR, p = 
0.004; two-sided independent sample t-test) 2. In our previous cross-sectional comparison,
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using the mean and standard deviations between lean and obese individuals, we expect an 
effect size of d=2.20 and with a power of 0.90, to account for possible smaller differences in 
males, we need 6 individuals per group. For reference, d=2.20 and 0.80 power would be 5 per 
group.
MMT: Postprandial whole-body energy expenditure per fat free mass (FFM) differences were 
shown for low-BAT (32±2 kcal/day/kg FFM) and high-BAT (37±2 kcal/day/kg FFM) 45. Based 
on these data, we can calculate an effect size of d=2.50. With a power of 0.80 required, we 
will need 8 individuals (4 per group) determine EE differences between low- and high-BAT 
individuals. Increasing the power to 0.95 will increased the number of individuals to 12 (6 per 
group). Due to the technical challenges of performing microneurography on multiple occasions 
in obese women, we overestimate and account for ~ 10% failure rate, so we will recruit 10 
subjects per group.
6.5
Planned Analyses
6.5.1
Primary Objective Analysis
The primary objective of this study is to quantify sympathetic nervous system (SNS) activity 
by [11C]MRB PET imaging and microneurography (MSNA). [11C]MRB binding will be quantified 
in adipose tissue (WAT and BAT), muscle; and whether [11C]MRB binding and sympathetic 
bursts are altered in individuals with obesity in comparison to individuals with normal weight.
Statistical Analysis: Before statistical testing, all continuous variables will be tested for 
normality of distribution. Summary statistics will be computed. For between-group comparisons 
of patient characteristics, two sample t-test or Mann-Whitney U test will be used for continuous 
variables, and Fisher’s exact test for categorical variables. Comparisons of repeatedly 
measured MSNA will be made using the mixed model analysis. Fixed factors include group 
and sex and their interactions. Linear contrasts comparing differences between groups or 
different time points within a group will be estimated.
6.5.2
Secondary Objectives Analyses
1) to evaluate whether gender differences affect peripheral SNS in healthy normal weight 
and obese men and women in adipose tissue (WAT and BAT) and muscle (resting/fasting);
2) To investigate the relationship between peripheral and central SNS activity (in obesity, by 
correlating SNS activity in peripheral tissues (WAT, BAT, and muscle) and brain; and 3) To 
investigate NET CNS and peripheral SNS activity before and after a high carbohydrate 
mixed meal in lean and obese men and women.
6.5.3
Exploratory Objectives Analyses (if applicable)
N/A
6.5.4
Safety
The principal investigator Renata Belfort De Aguiar, MD will be responsible for monitoring 
the data, assuring protocol compliance, and conducting the safety reviews at the specified
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frequency, which must be conducted at a minimum of every 6 months (including when 
reapproval of the protocol is sought). During the review process, the principal investigator 
will evaluate whether the study should continue unchanged, require 
modification/amendment, or close to enrollment. Either the principal investigator, the HIC, 
the NIH (NIDA), or the RSC have the authority to stop or suspend the study or require 
modifications.
6.5.5
Analysis of Subject Characteristics
The goal of this study is to measure in 20 individuals with normal weight (BMI 18-25 kg/m2) 
and 20 individuals with obesity (BMI 30-50 kg/m2)
6.5.6
Interim Analysis (if applicable)
N/A
6.5.7
Health economic evaluation
Subjects will paid $25 for the screening OGTT visit, regardless of the outcome, $200 for 
completion of each PET scan sessions (2 in total, $400), $100 for completion of each SNSA 
visit (2 in total, $200), $25 for each Mixed Meal Test (2 in total, $50),
and $100 bonus for completion of all 5 visits.
Subjects will be sent a Bank of America reloadable card that will be sent to their confirmed 
home address upon completion of each study visit as detailed above. In addition, subjects 
will be provided with a light lunch on PET day.
2. Costs for Participation (Economic Considerations): Clearly describe the subject’s costs 
associated with participation in the research, and the interventions or procedures of the 
study that will be provided at no cost to subjects.
No cost to subject related to participation in this research study.
3. In Case of Injury: This section is required for any research involving more than minimal 
risk, and for minimal risk research that presents the potential for physical harm (e.g., 
research involving blood draws).
a.
Will medical treatment be available if research-related injury occurs? Yes
b.
Where and from whom may treatment be obtained? Subjects would be assessed by 
PET Center practitioners and referred as appropriate to medical services.
c.
Are there any limits to the treatment being provided? No
d.
Who will pay for this treatment? Subject or subject’s insurance
e.
How will the medical treatment be accessed by subjects? Subjects would be 
assessed by PET Center practitioners and referred as appropriate to medical services.
Medical treatment will be offered to the subjects for any physical injuries that they receive as 
a result of participating in this research. However, the subject or his/her insurance company 
is responsible for the cost. Federal regulations require that subjects be told that if they are 
physically injured, no additional financial compensation is available.
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6.5.8
Other
N/A
6.5.9
Subsets and Covariates
N/A
6.5.10 Handling of Missing Data
Analyses will be conducted on available data. Adjustments to statistical models will be 
performed, as appropriate and necessary.
7
Trial Administration
7.1
Ethical Considerations: Informed Consent/Assent and HIPAA Authorization
7.2
Institutional Review Board (IRB) Review
The protocol will be submitted to the IRB for review and approval. Approval of the protocol 
must be obtained before initiating any research activity. Any change to the protocol or study 
team will require an approved IRB amendment before implementation. The IRB will 
determine whether informed consent and HIPAA authorization are required.
The IRB will conduct continuing review at intervals appropriate to the degree of risk, but not 
less than once per year.
A study closure report will be submitted to the IRB after all research activities have been 
completed.
Other study events (e.g. data breaches, protocol deviations) will be submitted per [insert 
institution's] IRB's policies.
7.3
Subject Confidentiality
Required private identifiable information about individuals, such as their medical history, 
current medications, psychiatric problems, and family history, will be collected by research 
staff and be used for research purposes and charting after consent is obtained.
HIPAA identifiers:
  Telephone numbers 
 E-mail addresses
 Medical record numbers
  All elements of dates (except year) for dates related to an individual, including: 
birth date, admission date, discharge date, date of death, all ages over 89 and all 
elements of dates (including year) indicative of such age, except that such ages and 
elements may be aggregated into a single category of age 90 or older
The data are collected and recorded by trained research personnel. The data will be 
recorded in Excel spreadsheets that will be saved onto a University encrypted computer or a
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secure server, or will be in the form of questionnaires that are filled out by the subject or the 
researcher. These paper research materials containing confidential information are stored in 
locked filing cabinets. Additional brain data is collected during the brain imaging scans by 
trained technologists and is stored on password-protected and encrypted computers with 
identifying information carefully in compliance with HIPAA regulations.
This research is covered by a Certificate of Confidentiality from the National Institutes of 
Health. The researchers with this Certificate may not disclose or use information, 
documents, or biospecimens that may identify you in any federal, state, or local civil, 
criminal, administrative, legislative, or other action, suit, or proceeding, or be used as 
evidence, for example, if there is a court subpoena, unless you have consented for this use. 
Information, documents, or biospecimens protected by this Certificate cannot be disclosed to 
anyone else who is not connected with the research except, if there is a federal, state, or 
local law that requires disclosure (such as to report child abuse or communicable diseases 
but not for federal, state, or local civil, criminal, administrative, legislative, or other 
proceedings, see below); if you have consented to the disclosure, including for your medical 
treatment; or if it is used for other scientific research, as allowed by federal regulations 
protecting research subjects
7.4
Deviations/Unanticipated Problems
If the study team becomes aware of an anticipated problem (e.g. data breach, protocol 
deviation), the event will be reported to the IRB.
7.5
Data Collection
All digital data will be stored either on a secured server or on University encrypted desktops 
or laptops. All staff members that come into contact with the data are fully trained to the current 
HIPAA regulations and are informed as to the proper use of all data. Identifiable paper 
information is kept in locked file drawers and password protected computer files. Results are 
published as group data without the use of characteristics that would identify individual 
subjects. We quote information only by number in conference discussions, scientific reports, 
or publications, in order to maintain anonymity. Identifiable research data, including 
recruitment and screening information and code keys, are stored on a secure database 
located on the internal PET Center Network. The PET network is protected by a Cisco PIX 
firewall operated by ITS. All research data are backed up nightly to a Dell PV-136T library wit 
4 IBM Ultrium-TD2 tape drives using the backup software Legato Networker 7.3 from EMC. 
Human subjects enrolled in the study are assigned a subject-specific random identifier. 
Subject identifiers and the means to link the subject names and codes with the research data 
are stored in separate locations within the database. The software of the database limits the 
ability to connect the random identifier to the actual subject identification information to 
research team members only. Access to the database is password protected and each 
research team member is required to have a unique ID and password to gain access to the 
database. Authorized users employ their netid and authentication is performed using Yale’s 
central authentication server. Users always access research data through the random 
identifier only. Direct identifiers belonging to subjects who withdraw from the study, will be 
stripped from the key.
The data will be stored in locked filing cabinets and on the password-protected secure 
database on the internal Yale University PET Center Network for at least 7 years after the 
close of the study, accessed only by authorized personnel. After 7 years, the data will be de- 
identified. Data may be kept indefinitely.
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7.6
Data Quality Assurance
See 6.5.4
7.7
Study Records

Regulatory Documents

EKG 

Consent Forms

Subject Medical Records

PET imaging data (and related scan day paperwork)

Biospecimens (and related lab request paperwork – e.g. blood insulin 
measurements)
7.8
Access to Source Documents
See 7.5
7.9
Data or Specimen Storage/Security
See 7.5
7.10 Retention of Records
The data will be stored in locked filing cabinets and on the password-protected secure 
database on the internal Yale University PET Center Network for at least 7 years after the 
close of the study, accessed only by authorized personnel. After 7 years, the data will be de- 
identified. Data may be kept indefinitely.
7.11 Study Monitoring
The principal investigator Renata Belfort De Aguiar will be responsible for monitoring the 
data, assuring protocol compliance, and conducting the safety reviews at the specified 
frequency, which must be conducted at a minimum of every 6 months (including when 
reapproval of the protocol is sought). During the review process, the principal investigator 
will evaluate whether the study should continue unchanged, require 
modification/amendment, or close to enrollment. Either the principal investigator, the HIC, 
the NIH (NIDA), or the RIDC have the authority to stop or suspend the study or require 
modifications.
7.12 Data Safety Monitoring Plan
The principal investigator Dr. Belfort De Aguiar. will conduct a review of all adverse events 
upon completion of every study subject. The principal investigator will evaluate the frequency 
and severity of the adverse events and determine if modifications to the protocol or consent 
form are required.
Research staff will call subject 1-3 days after the PET scan to check in and address any 
adverse events that may have arisen post PET scan.
The study will be stopped if either of the following occur:
1) If 1 or more subjects experiences a serious adverse event (SAE), which is considered by 
the investigator to be possibly or probably related to the radiotracer.
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2) If 2 or more subjects experience AEs of severe intensity, which are considered by the 
investigator to be possibly or probably related to the radiotracer and of clinical concern.
After review of the safety data, SAEs and AEs confirmed to have been related to the 
radiotracer will be reported to the FDA and the appropriate regulatory committees at Yale. 
The study will be placed on hold until a determination is made as to whether the study may 
continue as written, if modifications are needed, or if the study will be discontinued.
7.13 Study Modification
Study modifications will be submitted to the IRB review and subjects will be contacted if 
necessary.
7.14 Study Discontinuation
If the study is prematurely terminated or suspended, the Principal Investigator (PI) will 
promptly inform study participants, the Institutional Review Board (IRB), and sponsor and will 
provide the reason(s) for the termination or suspension. Study participants will be contacted, 
as applicable, and be informed of changes to study visit schedule.
Circumstances that may warrant termination or suspension include, but are not limited to:

Determination of unexpected, significant, or unacceptable risk to participants

Demonstration of efficacy that would warrant stopping

Insufficient compliance to protocol requirements

Data that are not sufficiently complete and/or evaluable

Determination that the primary endpoint has been met

Determination of futility
7.15 Study Completion
When participants have completed study and analysis has been complete, or if any other 
factor would lead to the shutdown of the study i.e. loss of funding or safety concerns, the 
IRB will be notified via their respective reporting channel..
7.16 Conflict of Interest Policy
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who 
have a role in the design, conduct, analysis, publication, or any aspect of this trial will be 
disclosed and managed. Furthermore, persons who have a perceived conflict of interest will 
be required to have such conflicts managed in a way that is appropriate to their participation 
in the trial. The study leadership in conjunction with the appropriate conflict of interest review 
committee has established policies and procedures for all study group members to disclose 
all conflicts of interest and will establish a mechanism for the management of all reported 
dualities of interest.
All investigators will follow the applicable conflict of interest policies.
7.17 Funding Source
NIH/NIDDK 1R56DK129344-01
7.18 Publication Plan
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These studies are likely to produce novel results relating to obesity and SNS that will merit 
publication, and the PI (Renata Belfort De Aguiar, MD, PhD) will be responsible for 
publishing any manuscripts resulting from these efforts.
8 Appendices
Appendix #
Title
Section
Topic
9 List of Tables
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10 References:
1.
Lin KS, Ding YS. Synthesis, enantiomeric resolution, and selective C-11 methylation of a 
highly selective radioligand for imaging the norepinephrine transporter with positron emission 
tomography. Chirality 2004;16:475-81.
2.
Sanchez-Rangel E, Gallezot JD, Yeckel CW, et al. Norepinephrine transporter availability in 
brown fat is reduced in obesity: a human PET study with [(11)C] MRB. Int J Obes (Lond) 2019.
3.
Innis RB, Cunningham VJ, Delforge J, et al. Consensus nomenclature for in vivo imaging of 
reversibly binding radioligands. J Cereb Blood Flow Metab 2007;27:1533-9.
4.
Ichise M, Liow JS, Lu JQ, et al. Linearized reference tissue parametric imaging methods: 
application to [11C]DASB positron emission tomography studies of the serotonin transporter in human 
brain. J Cereb Blood Flow Metab 2003;23:1096-112.
5.
Hannestad J, Gallezot JD, Planeta-Wilson B, et al. Clinically Relevant Doses of 
Methylphenidate Significantly Occupy Norepinephrine Transporters in Humans In Vivo. Biol 
Psychiatry 2010.
6.
Schou M, Halldin C, Pike VW, et al. Post-mortem human brain autoradiography of the 
norepinephrine transporter using (S,S)-[18F]FMeNER-D2. Eur Neuropsychopharmacol 2005;15:517- 
20.
7.
Ghazi T, Rink L, Sherr JL, Herold KC. Acute metabolic effects of exenatide in patients with 
type 1 diabetes with and without residual insulin to oral and intravenous glucose challenges. Diabetes 
Care 2014;37:210-6.
8.
Cersosimo E, Solis-Herrera C, Trautmann ME, Malloy J, Triplitt CL. Assessment of pancreatic 
beta-cell function: review of methods and clinical applications. Curr Diabetes Rev 2014;10:2-42.
9.
Maki KC, McKenney JM, Farmer MV, Reeves MS, Dicklin MR. Indices of insulin sensitivity 
and secretion from a standard liquid meal test in subjects with type 2 diabetes, impaired or normal 
fasting glucose. Nutr J 2009;8:22.
10.
Wallin BG, Charkoudian N. Sympathetic neural control of integrated cardiovascular function: 
insights from measurement of human sympathetic nerve activity. Muscle Nerve 2007;36:595-614.
11.
Tan CL, Knight ZA. Regulation of Body Temperature by the Nervous System. Neuron 
2018;98:31-48.
12.
Hwang JJ, Yeckel CW, Gallezot JD, et al. Imaging human brown adipose tissue under room 
temperature conditions with (11)C-MRB, a selective norepinephrine transporter PET ligand. 
Metabolism 2015;64:747-55.
13.
Casa DJ, Becker SM, Ganio MS, et al. Validity of devices that assess body temperature 
during outdoor exercise in the heat. J Athl Train 2007;42:333-42.
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14.
Ganio MS, Brown CM, Casa DJ, et al. Validity and reliability of devices that assess body 
temperature during indoor exercise in the heat. J Athl Train 2009;44:124-35.
15.
Emdin M, Gastaldelli A, Muscelli E, et al. Hyperinsulinemia and autonomic nervous system 
dysfunction in obesity: effects of weight loss. Circulation 2001;103:513-9.
16.
Straznicky NE, Lambert GW, Masuo K, et al. Blunted sympathetic neural response to oral 
glucose in obese subjects with the insulin-resistant metabolic syndrome. Am J Clin Nutr 2009;89:27- 
36.
17.
Scherrer U, Randin D, Tappy L, Vollenweider P, Jequier E, Nicod P. Body fat and sympathetic 
nerve activity in healthy subjects. Circulation 1994;89:2634-40.
18.
Grassi G, Seravalle G, Cattaneo BM, et al. Sympathetic activation in obese normotensive 
subjects. Hypertension 1995;25:560-3.
19.
Carnagarin R, Matthews VB, Herat LY, Ho JK, Schlaich MP. Autonomic Regulation of 
Glucose Homeostasis: a Specific Role for Sympathetic Nervous System Activation. Curr Diab Rep 
2018;18:107.
20.
Thorp AA, Schlaich MP. Relevance of Sympathetic Nervous System Activation in Obesity and 
Metabolic Syndrome. J Diabetes Res 2015;2015:341583.
21.
Esler M, Straznicky N, Eikelis N, Masuo K, Lambert G, Lambert E. Mechanisms of 
sympathetic activation in obesity-related hypertension. Hypertension 2006;48:787-96.
22.
Szabadi E. Functional neuroanatomy of the central noradrenergic system. J 
Psychopharmacol 2013;27:659-93.
23.
Ding YS, Lin KS, Garza V, et al. Evaluation of a new norepinephrine transporter PET ligand in 
baboons, both in brain and peripheral organs. Synapse 2003;50:345-52.
24.
Lin SF, Fan X, Yeckel CW, et al. Ex vivo and in vivo evaluation of the norepinephrine 
transporter ligand [11C]MRB for brown adipose tissue imaging. Nucl Med Biol 2012;39:1081-6.
25.
Belfort-DeAguiar RD, Gallezot JD, Hwang JJ, et al. Noradrenergic Activity in the Human 
Brain: A Mechanism Supporting the Defense Against Hypoglycemia. J Clin Endocrinol Metab 2018.
26.
Li CS, Potenza MN, Lee DE, et al. Decreased norepinephrine transporter availability in 
obesity: Positron Emission Tomography imaging with (S,S)-[(11)C]O-methylreboxetine. Neuroimage 
2014;86:306-10.
27.
Spraul M, Anderson EA, Bogardus C, Ravussin E. Muscle sympathetic nerve activity in 
response to glucose ingestion. Impact of plasma insulin and body fat. Diabetes 1994;43:191-6.
28.
Yeckel CW, Gulanski B, Zgorski ML, Dziura J, Parish R, Sherwin RS. Simple exercise 
recovery index for sympathetic overactivity is linked to insulin resistance. Med Sci Sports Exerc 
2009;41:505-15.
29.
Zygmunt A, Stanczyk J. Methods of evaluation of autonomic nervous system function. Arch 
Med Sci 2010;6:11-8.
30.
Usselman CW, Adler TE, Leone C, Paidas MJ, Stachenfeld NS. Sympathetic Nervous System 
Reactivity in Women following Preeclamptic Pregnancies. The FASEB Journal 2018;32:714.7-.7.
31.
Steffens AB, Damsma G, van der Gugten J, Luiten PG. Circulating free fatty acids, insulin, 
and glucose during chemical stimulation of hypothalamus in rats. Am J Physiol 1984;247:E765-71.
32.
Cincotta AH, Luo S, Zhang Y, et al. Chronic infusion of norepinephrine into the VMH of normal 
rats induces the obese glucose-intolerant state. Am J Physiol Regul Integr Comp Physiol 
2000;278:R435-44.
33.
Shimazu T, Noma M, Saito M. Chronic infusion of norepinephrine into the ventromedial 
hypothalamus induces obesity in rats. Brain Res 1986;369:215-23.
34.
Hesse S, Becker GA, Rullmann M, et al. Central noradrenaline transporter availability in 
highly obese, non-depressed individuals. Eur J Nucl Med Mol Imaging 2017;44:1056-64.
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35.
Vettermann FJ, Rullmann M, Becker GA, et al. Noradrenaline transporter availability on 
[(11)C]MRB PET predicts weight loss success in highly obese adults. Eur J Nucl Med Mol Imaging 
2018;45:1618-25.
36.
Reddy DS. Current pharmacotherapy of attention deficit hyperactivity disorder. Drugs Today 
(Barc) 2013;49:647-65.
37.
Brennan AR, Arnsten AF. Neuronal mechanisms underlying attention deficit hyperactivity 
disorder: the influence of arousal on prefrontal cortical function. Ann N Y Acad Sci 2008;1129:236-45.
38.
Glick Z, Raum WJ. Norepinephrine turnover in brown adipose tissue is stimulated by a single 
meal. Am J Physiol 1986;251:R13-7.
39.
Cox HS, Kaye DM, Thompson JM, et al. Regional sympathetic nervous activation after a large 
meal in humans. Clin Sci (Lond) 1995;89:145-54.
40.
Chen PC, Chan YT, Chen HF, Ko MC, Li CY. Population-based cohort analyses of the 
bidirectional relationship between type 2 diabetes and depression. Diabetes Care 2013;36:376-82.
41.
Fagius J, Berne C. Increase in muscle nerve sympathetic activity in humans after food intake. 
Clin Sci (Lond) 1994;86:159-67.
42.
Scott EM, Greenwood JP, Vacca G, Stoker JB, Gilbey SG, Mary DA. Carbohydrate ingestion, 
with transient endogenous insulinaemia, produces both sympathetic activation and vasodilatation in 
normal humans. Clin Sci (Lond) 2002;102:523-9.
43.
Berne C, Fagius J, Niklasson F. Sympathetic response to oral carbohydrate administration. 
Evidence from microelectrode nerve recordings. J Clin Invest 1989;84:1403-9.
44.
Saito M, Matsushita M, Yoneshiro T, Okamatsu-Ogura Y. Brown Adipose Tissue, Diet- 
Induced Thermogenesis, and Thermogenic Food Ingredients: From Mice to Men. Front Endocrinol 
(Lausanne) 2020;11:222.
45.
M UD, Saari T, Raiko J, et al. Postprandial Oxidative Metabolism of Human Brown Fat 
Indicates Thermogenesis. Cell Metab 2018;28:207-16 e3.
46.
Flegal KM, Kruszon-Moran D, Carroll MD, Fryar CD, Ogden CL. Trends in Obesity Among 
Adults in the United States, 2005 to 2014. JAMA 2016;315:2284-91.
47.
Ogden CL, Yanovski SZ, Carroll MD, Flegal KM. The epidemiology of obesity. 
Gastroenterology 2007;132:2087-102.
48.
Bray GA, York DA. Hypothalamic and genetic obesity in experimental animals: an autonomic 
and endocrine hypothesis. Physiol Rev 1979;59:719-809.
49.
Young JB, Macdonald IA. Sympathoadrenal activity in human obesity: heterogeneity of 
findings since 1980. Int J Obes Relat Metab Disord 1992;16:959-67.
50.
Guarino D, Nannipieri M, Iervasi G, Taddei S, Bruno RM. The Role of the Autonomic Nervous 
System in the Pathophysiology of Obesity. Front Physiol 2017;8:665.
51.
Funder JW, Carey RM, Fardella C, et al. Case detection, diagnosis, and treatment of patients 
with primary aldosteronism: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 
2008;93:3266-81.
52.
Vollenweider P, Randin D, Tappy L, Jequier E, Nicod P, Scherrer U. Impaired insulin-induced 
sympathetic neural activation and vasodilation in skeletal muscle in obese humans. J Clin Invest 
1994;93:2365-71.
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