Beige Fat, Energy, and the Natriuretic Peptide System (ENDA-025-17S)
Conditions: Obesity
Sponsor: VA Office of Research and Development

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Protocol Date: 07/21/2023 
 
 
 
 
 
 
 
 
 
Beige Fat, Energy, and the Natriuretic Peptide 
System 
 
 
Talat Alp Ikizler, M.D. FASN 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Protocol Date: 07/21/2023 
 
Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Statistical Considerations 
11.0 Privacy/Confidentiality Issues 
12.0 Follow-up and Record Retention 
 
 

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Protocol Date: 07/21/2023 
 
1.0 
Background 
 
Obesity and obesity-related complications are leading causes of morbidity 
and mortality among Veterans. Obese individuals experience a markedly increased 
risk of cardiovascular and metabolic (“cardiometabolic”) diseases– including diabetes, 
hypertension, and coronary artery disease. The rising prevalence of these problems 
despite current therapies supports that obesity is a multifactorial disease and highlights 
the need for novel approaches to treat obesity-related cardiometabolic dysfunction. 
Evidence from genetic studies in animals and humans indicate that the 
natriuretic peptide (NP) system may protect against cardiometabolic risk. The 
NPs are cardiac derived hormones, classically recognized for their role in the regulation 
of blood pressure and volume status. In recent years, animals studies and human 
genetic investigation have demonstrated that higher genetically-determined NP levels 
protect against  development of hypertension, obesity, Type 2 diabetes, and metabolic 
syndrome.1, 2 Further, administration of B-type natriuretic peptide (BNP) to wild-type 
mice leads to increased thermogenic gene expression in brown as well as white adipose 
depots, increased energy expenditure, and reduced fat accumulation.3 The increase in 
thermogenic gene expression in white adipose tissue reflects the development of 
“brown-like” or “beige” adipose tissue (a process referred to as “beiging”). Brown and 
beige adipose tissue increase energy expenditure by dissipating energy as heat 
(thermogenesis) through the actions of uncoupling protein 1 (UCP1).4  Promoting the 
development of a beige adipose phenotype could potentially lead to increases in energy 
expenditure, promote weight loss, and improve insulin sensitivity in obesity. 
NP levels are lower in obese compared with lean individuals,5 and in black 
compared with white individuals,6, 7 in human epidemiologic investigations. 
This suggests that low circulating NP levels may reflect a relative NP deficiency in obese 
and black individuals. As the NP system appears to protect against cardiometabolic risk, 
a relative NP deficiency could contribute to adverse cardiometabolic outcomes observed 
in obese and black individuals. Whether a relative NP deficiency in these groups is 
associated with reduced thermogenic gene expression and lower energy expenditure is 
not well understood. 
 
 
2.0 
Rationale and Specific Aims 
 
NP levels differ by obesity status and race in epidemiologic studies, and NPs 
appear to promote “beiging” of adipose tissue in animals. However, whether 
gene expression of beige adipose tissue differs by obesity status and race has 
not been established in humans. Human mechanistic studies investigating the 
relationships of beige adipose tissue markers with obesity status, race, and the 
natriuretic peptide system are limited. We propose an innovative physiologic study in 
which we will quantify adipose tissue gene expression and energy expenditure in states 
of relative NP deficiency in humans, namely in obese and black individuals. Our 
overarching postulate is that obese and black individuals have NP deficiencies that 
contribute to less beige adipose tissue and lower energy expenditure.  Thus, we propose 
the following aims: 
 
 
 

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Aim 1: Determine whether beige adipose tissue markers differ between 
obese and lean individuals. 
We plan to enroll and conduct subcutaneous adipose biopsies on approximately 155 
adult participants (aged 18-55 years; approximately 50 lean and 105 obese 
participants).  We will quantify expression of genes indicative of “beiging” in 
subcutaneous adipose tissue biopsies and determine whether gene expression differs 
based on obesity status. In a secondary analysis, we will determine whether these 
beige adipose tissue markers differ by race.  
 
Aim 2: Determine whether beige adipose tissue markers and energy 
expenditure are associated with natriuretic peptide receptor expression in 
adipose tissue and circulating natriuretic peptide levels. We will examine these 
associations in approximately 155 adult participants, as described in Aim 1.  
Secondary Aims:  
-Determine whether adipose tissue gene expression profiles suggestive of beiging 
differ by race.  
-Determine whether obesity-related differences in beige fat are attributable to 
differences in NP markers. 
  -Examine associations of adipose tissue phenotypes with cardiometabolic parameters. 
 
 
3.0 
Animal Studies and Previous Human Studies 
 
The natriuretic peptide (NP) system and metabolism 
 
The NP system is classically known for its role regulating blood pressure and 
volume status. In response to increased cardiac wall stress, the heart secretes atrial 
natriuretic peptide (ANP) and BNP, which both cause natriuresis, vasodilation, and 
inhibition of the renin-angiotensin-aldosterone system. Thus, the biologic actions of the 
NPs serve to reduce blood pressure. Both ANP and BNP bind to their target receptor, 
NPR-A, and are cleared by the clearance receptor, NPR-C.1 NP receptors are widely 
present throughout the cardiovascular system, as well as in adipose tissue, skeletal 
muscle, liver, brain, and gut.  
Although the NP system is classically known for its role in blood pressure 
regulation, recent studies have shown that NPs have important metabolic 
actions as well. Administration of NPs stimulate lipolysis in animals and humans.8-11 
Transgenic mice that overexpress BNP are protected against weight gain, visceral fat 
accumulation, and insulin resistance induced by high-fat diets.1 Moreover, Bordiccia et 
al. demonstrated in wild-type mice that BNP administration over 7 days significantly 
increases energy expenditure and oxygen consumption.3 The increases in energy 
expenditure and oxygen consumption appear to be mediated by increased expression of 
thermogenic genes (including UCP1) in brown adipose tissue, as well as white adipose 
tissue.3 The increase in thermogenic gene expression in white adipose tissue reflects the 
development of beige fat. 
 
 
 
 
The NP system, obesity, and cardiometabolic risk 

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Protocol Version #: 12 
 
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The NP system may protect against cardiometabolic risk. In humans, a common 
genetic variant resulting in ~10-20% higher NP concentrations (still within the 
physiologic range) is associated with lower prevalence of hypertension (by 15%),2 
obesity (by 46%), and metabolic syndrome (by 42%), and a lower risk of developing 
Type 2 diabetes (by 12%).12, 13 Moreover, this genetic variant that causes higher NP 
levels is associated with favorable cardiovascular biomarkers and a lower prevalence of 
myocardial infarction.12 Conversely, humans with a genetic variant leading to lower NP 
levels exhibit higher risk of hypertension, future T2DM,12, 13 and evidence of adverse 
cardiac remodeling.14 These data strongly suggest that the NP system may protect 
against cardiometabolic risk. 
 
Obese and black individuals have low circulating NP levels,5 which appears to 
reflect a relative “NP deficiency.” Multiple large cohort studies have demonstrated 
that NP levels are lower in obese compared with lean individuals,5 and in black 
compared with white individuals.6, 7 Obese and black individuals have a propensity 
toward hypertension, which would be expected to cause higher circulating NP 
concentrations. Thus, the lower NP concentrations in obese and black individuals are 
physiologically inappropriate and likely reflect a relative NP deficiency. As the NP system 
appears to protect against cardiometabolic risk, a relative NP deficiency could raise 
susceptibility to the adverse cardiometabolic outcomes observed in obese and black 
individuals. The reasons for the low NP levels in obese and black individuals are not 
entirely known.  One proposed mechanism for the low NP concentrations in obesity is 
enhanced NP clearance in adipose tissue. Obese individuals have been found to have 
higher expression of the NP clearance receptor in adipose tissue compared with lean 
individuals.15 Despite these associations, thermogenic gene expression in adipose tissue 
and whole body energy expenditure in these relatively NP deficient groups are not well 
characterized and understood. 
 
 
4.0 
Inclusion/Exclusion Criteria 
 
We will enroll and conduct subcutaneous adipose biopsies on approximately 155 
participants (approximately 50 lean subjects (18.5≤BMI<25 kg/m2) and approximately 
105 obese subjects (BMI≥30 kg/m2)). Full eligibility criteria are listed below. 
 
Inclusion Criteria:  
• 
Men and women ages 18-55 years  
• 
Body Mass Index (BMI) ≥18.5 and <25 kg/m2 (lean), or BMI ≥30 kg/m2 
(obese) 
 
Exclusion Criteria:  
• 
Significant pulmonary, liver, or renal disease 
• 
Heart failure (any type), unstable coronary artery disease currently, or history 
of significant cardiac event or stroke within 12 months 
• 
Diabetes Mellitus (Types 1 and 2) 
• 
Thyroid dysfunction 

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Protocol Date: 07/21/2023 
 
• 
Active malignancy 
• 
Chronic inflammatory diseases, such as inflammatory bowel disease, hepatitis, 
rheumatoid arthritis 
• 
Current use of medications likely to affect energy homeostasis, including 
amphetamines and beta blockers, and significant use of systemic 
glucocorticoids 
• 
Currently pregnant or breastfeeding, or unwilling to avoid becoming pregnant or 
breastfeeding during study duration 
• 
Significant claustrophobia that would prevent the use of the metabolic cart as 
part of the study protocol 
• 
Hemoglobin A1c (HbA1c) ≥6.5% 
• 
Liver Function Tests (LFTs) elevated >3x upper limit of normal 
• 
Estimated Glomerular Filtration Rate (eGFR) <40 ml/min 
 
 
5.0 
Enrollment/Randomization 
 
Recruitment will take place at Tennessee Valley Healthcare System (TVHS) outpatient 
clinics, community-based outpatient clinics, veteran centers, veteran service 
organizations, Fort Campbell, and Vanderbilt University Medical Center (VUMC).  All 
patients coming for outpatient services at TVHS may be considered. We will place IRB 
approved flyers and brochures in target clinics, veteran centers, Fort Campbell, and 
community boards (electronic and physical) with basic study information and study team 
contact information for potential subjects to signal potential interest. Working with the 
VA Public Affairs office, we will utilize their advertisement services such as waiting lobby 
TV advertisements, VA web homepage announcements, newspaper, and email 
distribution lists to veterans. A link may also be posted on My HealtheVet. We will also 
screen medical records in CPRS system (including data linked to CPRS through JLV and 
VISTA).  VA subjects, once identified through the CPRS system, will be sent recruitment 
letters about participation in the study. The recruitment letters may be followed by a 
phone call to the participant.  Study personnel may also approach possible study 
participants in clinic once permission is received by that subject’s provider. Providers and 
clinic teams members within the VA or non-VA medical centers may also send referrals 
to the study team. Subjects may also be recruited using My HealthEVet and the VINCI 
system. Subjects may also be recruited via the Vanderbilt Broadcast email system 
(research.notifications@vanderbilt.edu). ResearchMatch.org may be utilized as a 
recruitment tool for this protocol.  ResearchMatch.org is a national electronic, web-
based recruitment tool that was created through the Clinical & Translational Science 
Awards Consortium in 2009 and is maintained at Vanderbilt University as an IRB-
approved data repository. Study personnel may also use a VA-issued phone to 
communicate with recruited subjects (including phone calls and text messages for 
appointment reminders and logistical information). Text messages will not include any 
patient identifiers.  
 

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Protocol Date: 07/21/2023 
 
Due to recruitment setbacks related to COVID-19, some samples and data previously 
collected from another study, Cardiovascular Effects of GLP-1 Receptor Activation (P.I., 
Dr. J. Matthew Luther (Vanderbilt University Medical Center (VUMC) IRB# 170213, 
ClinicalTrials.gov Identifier: NCT03101930) will be included to help meet our enrollment 
goals for the obese subject population. In this prior study (VUMC IRB# 170213), obese 
individuals underwent the same study procedures that we are performing in the present 
study as outlined below (subcutaneous adipose tissue biopsy, indirect calorimetry, DXA 
body composition scan), and thus the samples/data have already been collected. These 
samples were of similar inclusion criteria as the currently recruiting study, with the 
minor adjustment of age criteria of up to 65 years of age. For the prior study (VUMC 
IRB#170213), subjects signed a consent form which indicated their consent for their 
samples/data to be used for future research. Samples provided from this study will be 
de-identified for our use. There are approximately 122 obese individuals from this prior 
study with available baseline samples/data relevant to any of our endpoints, of whom 
approximately 80 individuals have adipose biopsy data (our primary endpoint). The 
inclusion of these samples will enable us to analyze baseline data for approximately 105 
obese subjects with adipose biopsy data (primary endpoint) (from the subjects that we 
are able to personally recruit, as well as the previously collected samples/data from the 
prior study). 
 
 
6.0 
Study Procedures 
 
Screening Visit 
After informed consent has been obtained, the subject’s medical history and medications 
will be discussed and documented. A physical exam, including measurement of height, 
weight, and vital signs, will be performed.  Blood will be collected for a comprehensive 
metabolic panel, complete blood count (CBC), Hemoglobin A1c, thyroid stimulating 
hormone (TSH), and free T4. A urine or serum pregnancy test will be completed on 
female subjects of child-bearing potential. Whenever possible, we will combine these 
research screening tasks and procedures with standard of care tasks and procedures to 
decrease the burden placed on the patient. This information will be acceptable from 
standard of care visits as far back as 6 months prior to the day the patient is identified 
as a possible candidate. Even if the patient has had these assessments completed 
during a standard of care visit, it is up to the discretion of the Principal Investigator to 
use them or require an updated set of assessments. The Study Investigator may also 
request a second screening blood draw to confirm eligibility. Inclusion/exclusion criteria 
will be reviewed to confirm that the subject meets study eligibility requirements.  
Patients may be asked to stop spironolactone and eplerenone, if the participant is using 
them for cosmetic or excessive hair growth, 4-6 weeks prior to the study visit at 
Vanderbilt’s CRC, as these medications may affect the results of aldosterone and renin.   
 
Study Visit 
 
Subjects will arrive at the Vanderbilt Clinical Research Center (CRC) after having fasted 
for at least 8 hours. We will collect a urine sample, including urine sodium and 

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Protocol Date: 07/21/2023 
 
creatinine. Women of childbearing potential will undergo a pregnancy test. Subjects will 
then lie supine (flat on their back).  After approximately 30 minutes of lying supine, 
baseline resting energy expenditure will be assessed using indirect calorimetry 
(metabolic cart, described below).  At the completion of the metabolic cart, and while 
subject is still lying supine, we will collect a blood sample, including basic metabolic 
panel, measurements of NPs and measurements of the RAAS system (renin and 
aldosterone), insulin, glucose, and markers of lipolysis (glycerol, free fatty acids).  
Subjects will then undergo a subcutaneous fat biopsy, as described below.  
Subsequently gene expression will be assessed for markers of “beiging” (including 
UCP1), as well as other cardiometabolic pathways including the natriuretic peptide 
pathways. Protein expression and histology may also be assessed if sufficient tissue is 
obtained.  Subjects will also undergo a dual-energy x-ray absorptiometry (DXA) scan to 
assess body composition.  In the event of scheduling issues, the DXA scan may be 
performed within about 2 weeks before or after the main study visit. Subjects will need 
to remain lying supine for the majority of the study visit (about 2 hours, before and 
during the metabolic cart, blood draw, and fat biopsy).  
 
For women, these study visits will be scheduled to coincide with the follicular phase of 
the menstrual cycle to minimize the impact of hormonal changes on energy expenditure 
(EE) and on measurements of renin-angiotensin-aldosterone (RAAS) system.   
 
Other study procedures 
 
Phlebotomy: Blood draws will occur as outlined above. 
 
Subcutaneous adipose tissue biopsy: Participants will undergo percutaneous 
subcutaneous adipose tissue biopsies on the abdomen (peri-umbilical region). This 
procedure will be performed by a trained study physician or nurse practitioner. With 
the subject laying in a supine position, an approximately 10 cm diameter area will be 
cleaned with a chlorhexidine swab lateral to the umbilicus. A fenestrated sterile drape 
will be placed to establish the sterile field. Approximately 20 ml of 1% lidocaine will be 
applied to anesthetize the skin. A small (approximately 0.5 cm) incision is made 
through the skin with a scalpel, within a few centimeters lateral to the umbilicus. Upon 
examination of the participant’s body habitus, the trained proceduralist may choose to 
first infiltrate normal saline into the subcutaneous adipose tissue if the proceduralist 
feels that this may improve the success of obtaining sufficient adipose tissue. Then, a 
TULIP 2.7mm liposuction cannula with a syringe attached is inserted and moved 
parallel to the skin at a rate of approximately 1 Hz without breaking suction and with a 
twisting motion. The sampling continues until a goal of approximately 10 ml of tissue 
(approximately 9.5 g) is collected. The syringe is then removed, antibiotic ointment 
applied, a steri-strip is placed, and the incision is covered with an adhesive bandage. 
The tissue will be rinsed from blood. Then, tissue samples will either be placed 
immediately in liquid nitrogen and stored at -80 degrees Celsius until analysis, or 
processed immediately. The bandages will be removed after 24 hours, and steri-strips 
are left in place until they fall off. 
 
Indirect Calorimetry (Metabolic Cart): Resting energy expenditure will be determined by 
indirect calorimetry, using a metabolic cart (a ventilated hood system). 

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Hemodynamic monitoring (blood pressure monitoring and heart rate monitoring by 
cardiac telemetry): We will monitor heart rhythm and rate during the study. This will be 
done by placing sticky pads (electrodes) on the chest. The sticky pads are connected to 
wires (leads) that hook up to a machine and display heart rhythm, rate and blood 
pressure. 
 
Substrate oxidation (lipid oxidation and carbohydrate oxidation): Respiratory exchange 
ratio will be determined using VO2 and VCO2 measurements, which is determined via 
indirect calorimetry, and collected at the same time points as energy expenditure.  
 
Biomarkers: Blood, urine, and tissue samples will be coded for subject confidentiality.  
These samples will be measured for a wide array of cardiometabolic parameters and 
inflammatory markers. Possible techniques include RNAseq and metabolomics 
techniques. Additional samples will be frozen and stored for possible future 
investigation. 
 
Table Of Events:  
Procedure 
Screening Visit 
Main Study Visit 
Informed Consent 
X 
 
Medical History & 
Physical exam 
X 
 
Vital Signs, height, 
weight 
X 
X 
Pregnancy test 
X 
X 
Clinical Laboratory 
sample collection 
X 
X 
Fat Biopsy  
 
X 
Metabolic Cart 
 
X 
DXA scan 
 
X 
 
 
7.0 
Risks 
 
Fasting: Inconvenience and hypovolemia. To minimize risks of hypovolemia, subjects will 
be instructed to drink plenty of fluids the day before the visits, and we will measure 
subjects’ blood pressure and heart rate upon arrival for their study visits and during the 
study visits. Also, there is a potential risk of hypoglycemia while fasting. However, the 
risk of hypoglycemia in healthy individuals while fasting for this amount of time is low. 

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Protocol Version #: 12 
 
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Subjects will be monitored for signs and symptoms of hypoglycemia throughout the 
study visits. 
 
Phlebotomy: The risks associated with phlebotomy in healthy individuals are minimal. 
Risks include pain, bruising, inflammation, and rarely fainting and infection. Other 
possible risks include low fluid status in the body and anemia (low blood counts). Risks 
will be minimized by excluding subjects with anemia or history of hypotension. Subjects 
will be advised to drink plenty of the fluids the night before the study visit, and after the 
study visit.  
 
Hemodynamic monitoring (blood pressure monitoring and heart monitoring by cardiac 
telemetry): A non-invasive technique with no symptoms aside from possible minimal 
discomfort. 
 
Percutaneous subcutaneous adipose tissue biopsy: The biopsy procedure will be 
performed by a study physician or nurse practitioner trained in this technique. The 
percutaneous subcutaneous adipose tissue biopsy procedure is associated with low risk. 
Possible risks of the biopsy procedure include pain, local skin irritation, bleeding, 
bruising, and hematoma at the site. There is potential risk for local or systemic infection, 
more severe bleeding, or a small scar; however, the risk of these events is extremely 
low.  
Lidocaine: May cause local discomfort during injection, or a rash, redness or soreness at 
the injection site. In rare cases, lidocaine could potentially cause hypersensitivity 
reactions, confusion, or induce a transient alteration in heart rhythm; to reduce this risk, 
suction will be applied to the syringe prior to injection to ensure the lidocaine is not 
being injected into a vessel. 
Metabolic cart: This is a non-invasive technique which involves placing a ventilated hood 
over the subject’s head for approximately 30 minutes.  The technique carries no risks 
other than possible minimal discomfort.  Subjects with history of claustrophobia will be 
excluded from the study.  
Dual Energy X-ray Absorptiometry (DXA): Radiation risk is very minimal. DXA uses very 
low dose radiation, and the expected total exposure for each participant is in the range 
of 7 millirem, which is equivalent to approximately 9 days of background radiation.  
Subjects will be offered lunch after completing study procedures that must be performed 
in a fasted state (metabolic cart, blood draw, and abdominal fat biopsy). 
 
 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
 
Participants should and will be instructed to notify study personnel regarding any 
adverse events or unanticipated problems. Adverse events will be reported to the IRB 
per IRB policy. Also, all serious and unanticipated adverse events or problems involving 
risks to subjects that may possibly be or are known to be related to the research activity 
will be reported promptly to the IRB office per IRB policy.  
 

VA Tennessee Valley Healthcare System, 1310 24th Avenue South, Nashville, TN 37212 
Protocol Version #: 12 
 
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Protocol Date: 07/21/2023 
 
A Data Safety Monitor will monitor the study. The Data Safety Monitor will provide 
ongoing evaluation of studies’ progress including patient accrual and retention and 
monitoring of adverse events. Reports will be submitted to the Data Safety Monitor for 
review approximately every 12 months. 
 
 
 
9.0 
Study Withdrawal/Discontinuation 
 
Subjects may withdraw from the study at any time and should notify study personnel in 
writing if they wish to withdraw from the study. Subjects may request their biological 
samples to be destroyed at any time. However, any data or biological samples that have 
already been used for research cannot be destroyed.  
 
Subjects may be discontinued from the study at the discretion of the investigators’ 
(possible reasons listed below).  
 
Possible reasons for withdrawal/discontinuation from study include, but are not limited 
to: 
• 
Noncompliance with procedures 
• 
Decision by participant/participant withdraws consent 
• 
Loss to follow-up 
• 
Change in patient’s health status that makes them no longer eligible for study. 
• 
Development of a significant medical condition specified in the exclusion criteria 
• 
In female subjects, becoming pregnant during study 
• 
Significant adverse event deemed by investigator to preclude continued 
participation 
 
 
10.0 Statistical Considerations 
 
Sample size justification: A total of 100 subjects will provide 80% power to detect a 
minimum difference between lean and obese groups of 0.7 units of UCP1 mRNA 
expression normalized for housekeeping gene mRNA gene expression (primary analysis). 
This represents a metabolically meaningful difference of approximately 0.45 standard 
deviations between lean and obese groups. These calculations are based on a Type 1 
error rate of 0.05, and interindividual variations in UCP1 gene expression reported in 
prior studies.16  Since there is only 1 main study visit, dropouts should not be an issue in 
this study. However, there is a low likelihood that some adipose tissue samples may be 
inadequate quantity or quality.  Even if some adipose tissue samples are of insufficient 
quantity or quality, a sample size of 80 would still provide 80% power to detect a 
minimum difference between groups of 0.75 units in UCP1 normalized mRNA expression, 
which represents a metabolically meaningful difference of approximately 0.48 standard 
deviations between lean and obese groups. 

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Of note, the above sample size calculation was based on the original enrollment goal of 
100 subjects. Now that we have the benefit of including some samples from a previously 
completed study, our statistical power to answer our scientific questions will be 
improved.  
Statistical Analysis: The primary analysis will determine whether adipose tissue gene 
expression of UCP1 differs between lean and obese individuals. To assess this, we will 
use multivariable linear regression, with UCP1 gene expression as the outcome variable, 
and BMI as the independent variable. The multivariable linear regression will be 
adjusted for covariates that may impact markers of beige adipose tissue, including age 
and sex. The null hypothesis is that UCP1 gene expression will not differ based on BMI. 
Secondary analyses include whether UCP1 gene expression differs by race and whether 
expression of additional “beiging” markers differ by obesity status.  Like the primary 
analysis, the secondary analyses are analyzing a continuous outcome variable.  Thus, 
the statistical approach for the secondary analyses will be similar to that described for 
the primary analysis. 
 
 
11.0 Privacy/Confidentiality Issues 
 
Strict confidentiality will fully be maintained possible by the research team, including 
keeping all data in a secure location. All specimens will be de-identified and coded after 
they are obtained, and the code key kept in a secure location. Only study personnel will 
access to the data and specimens. Samples may be shared with third parties outside of 
the VA and Vanderbilt for future testing but will remain anonymous to the recipient.  
Subjects may contact the principal investigator at any time to request that samples be 
destroyed.  However, any data or biological samples that have already been used for 
research cannot be destroyed. 
 
The Research Electronic Data Capture (REDCap) database is a secure web application 
housed on the TVHS VA secured computer network and is password protected. REDCap 
offers secure institutional data hosting and includes full audit-trails in compliance with 
HIPAA security requirements. Only members of the study team will have access to this 
database.   
 
Adverse event reports and annual summaries will not include subject-identifiable 
material but only the assigned study identification numbers. 
 
 
12.0 Follow-up and Record Retention 
 
Anticipated study duration is 40 months. Research data will be maintained by the PI 
after study closure. After study closure, research data will be maintained for a minimum 
of 6 years and possibly indefinitely. Data will be stored on a secure computer network in 
a password-protected database. Only members of the study team will have access. 
Pertinent paper documentation will be kept in locked office and only study personnel will 
have access. Only personnel directly involved with the study will have access to source 
data and the electronic database. 
 

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