Assessments of Adipogenesis, Lipid Turnover and Cellular Composition in Adipose Tissue in Response to Endurance Exercise
Phase: NA
Conditions: Obesity
Sponsor: AdventHealth Translational Research Institute

 
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Study Protocol Title: 
Adipogenesis, triglyceride turnover and cellular composition of adipose tissue in response to 
endurance training (ATLAS). 
 
Study Sponsor:  
AdventHealth 
 
Funder:  
National Institutes of Health 
 
Principal Investigators: 
Lauren Sparks, PhD and Ursula White, PhD 
 
Medical Investigator: 
Richard Pratley, MD and Frank Greenway, MD 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Table of Contents  
List of Abbreviations ……………………………………………………………………………5 
Introduction ……….…………………………………………………………………………….6 
Background Information and Scientific Rationale ……………………………………………..6 
Study Objectives ………………………………………………………………………………..7 
Primary Objective/Aim/Goal/Hypothesis ………………………………………………………7 
Secondary Objective/Aim/Goal/Hypothesis ……………………………………………………7 
Study Design ……………………………………………………………………………………7 
Research Design ………………………………………………………………………………...7 
Research Intervention Description ……………………………………………………………...7 
Study Site(s)/Location(s) and Number of Subjects ……………………………………………..8 
Multi-Site Research Logistics/Communication Plan …………………………………………...8 
Research Conducted in a Foreign Country ……………………………………………………..9 
Community-Based Participatory Research ……………………………………………………..9 
Subject Selection ………………………………………………………………………………..9 
Vulnerable Populations ……………………………………………………………………...….9 
Inclusion Criteria ……………………………………………………………………………….10 
Exclusion Criteria ……………………………………………………………………………....10 
Resources Available ……………………………………………………………………………11 
Study Procedures ………………………………………………………………………………12 
Subject Recruitment and Screening ……………………………………………………………12 
Consent Process ………………………………………………………………………………..12 
Subjects who are not yet adults (infants, children, teenagers) …………………………………12 

 
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Cognitively Impaired Adults …………………………………………………………………12 
Adults Unable to Consent …………………………………………………………………….12 
Documentation of Informed Consent Process ………………………………………………..12 
Waiver of Written Documentation of Consent or Waiver of Consent ……………………….12 
Non-English Speaking Subjects ……………………………………………………………...13 
Randomization ……………………………………………………………………………….13 
Study Visits …………………………………………………………………………………..13 
Study Duration ……………………………………………………………………………….23 
Materials of Human Origin:  Collection, Preparation, Handling and Shipping ……………...23 
Study Outcome Measures (Endpoints) ……………………………………………………….24 
Data Management and Quality Plan ………………………………………………………….24 
Data De-identification ………………………………………………………………………..24 
Data Confidentiality, Storage, and Retention  ………………………………………………..25 
Data Quality ………………………………………………………………………………….25 
Data Sharing …………………………………………………………………………………26 
Sample Size Determination ………………………………………………………………….26 
Statistical Analysis Plan ……………………………………………………………………..27 
Primary and Secondary Objective Analysis …………………………………………………27 
Potential Benefits …………………………………………………………………………….27 
Potential Risks ……………………………………………………………………………….27 
Mitigation of Risks …………………………………………………………………………..28 
Provisions to Protect the Privacy Interest of Subjects ……………………………………….30 
Early Withdrawal of Subjects  ……………………………………………………………….30 

 
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Investigator Withdrawal of Subjects ……………………………………………………….  30 
Subject Request for Withdrawal from Study ………………………………………………..30 
Data Collection and Follow-up for Withdrawn Subjects ……………………………………30 
Adverse Events - Recording and Reporting …………………………………………………31 
Safety Monitoring Plan ……………………………………………………………………...31 
Safety Monitoring …………………………………………………………………………...31 
Data and Safety Monitoring Board (DSMB) or Equivalent ………………………………...31 
Ethical Considerations ………………………………………………………………………31 
Sharing of Results with Subjects ……………………………………………………………32 
Funding Source ……………………………………………………………………………..32 
Subject Stipends or Payments ………………………………………………………………32 
Publication Plan …………………………………………………………………………….33 
References ………………………………………………………………………………….34 
 
Appendix …………………………………………………………………………………...35 
 
 

 
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List of Abbreviations: 
 
ACSM – American College of Sports Medicine  
AH – AdventHealth  
BMI – Body Mass Index  
CRF – Case Report Forms  
DO – Doctor of Osteopathic Medicine  
ECG – Electrocardiogram  
EDC – Electronic Data Capture  
FDA - Food and Drug Administration  
GCP - Good Clinical Practices  
ICH - International Conference on Harmonization  
IR – Insulin Resistance  
IRB – Institutional Review Board  
MD – Medical Doctor  
MRI – Magnetic Resonance Imaging   
PBRC – Pennington Biomedical Research Center  
PCOS – Polycystic Ovarian Syndrome  
PHI – Protected Health Information  
PI – Principal Investigator  
PID – Participant Identifier  
RER – Respiratory Exchange Ratio  
SAE – Serious Adverse Event  
SOP –  Standard Operating Procedure  
TG – Triglyceride  
TRI - Translational Research Institute  
VO2 max – Maximal Volume of Oxygen Consumed  
WAT – White Adipose Tissue  
snRNAseq – single nuclei RNA sequencing  
 
  
  
 
 

 
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Introduction 
 
This document is a research protocol for a human research study to investigate the effects of 
exercise training on adipogenesis, triglyceride turnover and cellular composition of adipose 
tissue, and insulin resistance in subjects with obesity. The described study will be conducted in 
compliance with the research protocol, Good Clinical Practices (GCP) International Conference 
on Harmonization (ICH) Guidelines (E6) for GCPs standards as adopted by the Food and Drug 
Administration (FDA) and associated Federal regulations, and all applicable institutional 
research requirements.  
 
Background Information and Scientific Rationale 
 
Excess adiposity – obesity – causes insulin resistance (IR) in white adipose tissue (WAT) and 
other tissues.  The role of poor WAT quality in IR, however, is less clear. Low adipocyte 
triglyceride (TG) turnover, high formation rates of new adipocytes, high senescent cell burden, 
tissue and cellular inflammation and high circulating free fatty acids (FFAs) all characterize poor 
WAT quality [1, 2].   
 
Exercise generally improves whole-body IR and decreases fasting FFAs – even without 
reductions in overall fat mass – thus highlighting the significance of exercise effects on WAT 
quality. Evidence of the direct impact of exercise – independent of weight loss – on WAT-
specific IR is limited and inconclusive. The underlying mechanisms that drive exercise-induced 
changes in WAT biology, such as new adipocyte formation and TG turnover rates, cell 
composition, senescence, and inflammation, are poorly understood in humans.    
  
Using a suite of innovative methods, ours will be the first study to comprehensively assess the 
direct effects of exercise on features of WAT quality both in vivo (formation of new adipocytes 
and adipocyte TG turnover) and ex vivo (cellular landscape of WAT) and WAT-specific IR 
(suppression of lipolysis via subcutaneous microdialysis) in humans with obesity. Using a 
practical approach of isotopic labeling that incorporates deuterium (2H), administered as oral 
2H2O, into the DNA and TG moieties of adipocytes, we have compelling published and 
preliminary evidence that exercise reduces in vivo formation of new adipocytes in both mice and 
humans, suggesting an important exercise-induced mechanism of WAT remodeling [3-5]. We 
reason that increased adipocyte TG turnover rates improve adipocyte function, WAT-specific IR 
and overall WAT quality and thus abrogate the need for increased formation of new adipocytes.  
  
Adipocytes comprise 90% of the tissue volume, but only ~50% of WAT cellular content, with 
the remainder comprised of immune cells, stem cells, and pre-adipocytes [6, 7]. Using our 
established method [8], we will employ innovative single nuclei transcriptomics (snRNAseq) to 
discover the effects of exercise on senescence and inflammation within adipocytes and non-
adipocytes of WAT to further understand how exercise impacts WAT quality and WAT-specific 
IR via in vivo microdialysis during insulin suppression.    
  
 
 

 
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Study Objectives 
 
The overall aim of this study is to investigate the effects of exercise training on adipogenesis, TG 
turnover and cellular composition of adipose tissue, and insulin resistance in subjects with 
obesity.  
 
Primary Objective/Aim/Goal/Hypothesis 
 
The primary aim of this study is to investigate the effects of 12-weeks of endurance 
exercise training on in vivo formation of new adipocytes, TG turnover rates, and cellular 
composition of the WAT.  
  
Approach: A subcutaneous WAT biopsy from the abdomen will be collected before and 
after 12 weeks of deuterium (2H) labelled water administration and endurance exercise 
training in men and pre-menopausal women with obesity. Collected WAT samples will be 
utilized for in vivo measurements of adipocyte formation and TG turnover. The remaining 
WAT samples will be frozen for future analyses that include snRNAseq and histology to 
assess cellular composition, senescence, and inflammation of the WAT 
 
Secondary Objective/Aim/Goal/Hypothesis 
 
The secondary aim of this study is to assess the effects of 12-weeks of endurance exercise 
training on WAT-specific IR and fasting FFA levels and molecular adaptations in skeletal 
muscle that may contribute to the alterations in WAT.   
  
Approach: Blood samples and dialysates from abdominal subcutaneous WAT will be 
collected during hyperinsulinemic clamp to assess whole-body IR and WAT-specific 
suppression of lipolytic rates under physiological insulin in men and pre-menopausal 
women with obesity. A skeletal muscle biopsy from the vastus lateralis will be collected 
before and after 12 weeks of deuterium (2H) labelled water administration and endurance 
exercise training to assess transcriptomic and proteomic adaptation that may associate with 
WAT adaptations.   
 
Study Design 
 
Research Design 
 
This is a double-site (AH TRI and PBRC), longitudinal (12 weeks) randomized clinical 
trial with two groups; a control group (CTRL) and an exercise training group (EX) 
 
Research Intervention Description  
 
 

 
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Subjects will be submitted to 12 weeks of supervised aerobic exercise training 
intervention and oral deuterium-labelled water administration OR oral deuterium-labelled 
water administration only. 
 
This study consists of a Screening Period, Baseline Period, Intervention Period, 
and Follow-up Period, detailed below. 
 
Study Site(s)/Location(s) and Number of Subjects 
 
AdventHealth Orlando Sites: Translational Research Institute (TRI)  
  
Estimated number of subjects at AdventHealth Orlando site: 35 men and women 
(accounting for a 20% dropout; we expect 29 to complete the study)  
  
Name of external site(s) outside of AdventHealth Orlando: Pennington Biomedical 
Research Center   
  
Estimated number of subjects at external sites: 35 men and women (accounting for a 20% 
dropout; we expect 29 to complete the study)  
  
Total number of all sites: 2  
Estimated number of subjects at all sites combined: 70  
 
Multi-Site Research Logistics/Communication Plan 
 

 
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AdventHealth IRB will be the IRB of record for both sites, and AdventHealth will be the 
coordinating center. The two participating sites will meet via teleconference or phone call 
on a regular basis to discuss any issues, recruitment strategies, enrollment status, safety 
concerns, and any changes to the study protocol or supporting documents. Dr. Sparks and 
Dr. White have known each other for over a decade and have complementary skill sets in 
adipose tissue metabolism, as well as working closely together on the Program Planning 
Committee for The Obesity Society (TOS) for the past several years. Both investigators 
have done in-person site visits in the last 2 years in order to align their common laboratory 
practices related to this project, in addition to meeting virtually on a bi-weekly basis to plan 
this project. This project represents a continuation of the strong collaborations between AH 
TRI and PBRC. AH TRI and PBRC are both clinical sites in MoTrPAC (U01AR071133) 
and thus aligned and cross-validated for all clinical assessments, procedures, WAT 
processing and the exercise intervention. In broader terms, AH TRI and PBRC have long 
outstanding track records in leading multi-site lifestyle modification interventions in 
studies funded by the NIDDK (MoTrPAC, DPP, LookAHEAD), NIA (CALERIE 1 and 2, 
LIFE), and USDA (PROOF). While the proposed study will capitalize on our sites’ 
participations in MoTrPAC and align our overlapping clinical procedures and intervention 
accordingly, we cannot execute this as an ancillary to MoTrPAC due to the prospective 
nature of the 2H2O labeling, hyper-insulinemic clamp and large tissue requirements for the 
downstream assays. We have chosen to conduct this clinical exercise trial at two sites to 
maximize efficiency through use of common core capabilities and complementary bench-
based methods, to ensure successful completion of the enrollment goals, as well as enhance 
our ability to include African Americans (mainly from the PBRC population) and 
Hispanics (mainly from the AH TRI population).  
 
Research Conducted in a Foreign Country 
 
 
N/A 
 
Community-Based Participatory Research 
 
N/A 
 
Subject Selection 
 
Vulnerable Populations (if applicable) 
  
Cognitively Impaired Adults: N/A 
 
Children: N/A 
 
Pregnant Women: N/A 
 
Neonates of non-viable or uncertain viability: N/A 
 

 
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Prisoners: N/A 
 
Employees: Recruitment efforts will follow AdventHealth recruitment Standard 
Operating Procedures (SOPs) at AH or PBRC recruitment SOPs for research at 
PBRC. AdventHealth and PBRC employees will not be individually targeted nor 
excluded from study participation based on employment. AdventHealth and PBRC 
employees who engage the AdventHealth Translational Research Institute or PBRC 
asking to participate in the study will be processed per standard consent procedures for 
participants.  In addition, during the consent process, the study staff will review standard 
consent language stating that an employee’s participation or lack of participation in the 
study will not affect their employment status or relationship with AdventHealth or PBRC. 
 
Students: Students will not be individually targeted nor excluded from study 
participation based on employment.   
 
Inclusion Criteria 
 
1. Men and pre-menopausal women;   
2. 18-45 years of age;   
3. BMI 30.0-40.0 kg/m2;   
4. For women, if not using pharmaceutical (hormonal) contraception (i.e. 
birth control pills, vaginal ring, injections, or implant), must agree to use 
either a double barrier method as a form of birth control to prevent 
pregnancy (i.e. male condom with spermicide, with or without cervical 
cap or diaphragm); use implants or intrauterine contraceptive devices; 
have a tubal ligation (surgically sterile); practice abstinence; or be in an 
established relationship with a vasectomized or same sex partner during 
the entire duration of the study;   
5. Able to speak and understand written and spoken English; 
6. Sedentary defined as self-reporting no more than 1 day per week, lasting 
no more than 60 minutes, of regular (structured) endurance or resistance 
exercise in the past year. 
7. Must be willing to be randomized to either study group;   
8. Must be willing to adhere to all study procedures.   
9. Participant has voluntarily signed and dated an informed consent form, 
approved by an Institutional Review Board/Independent Ethics 
Committee, and provided Health Insurance Portability and Accountability 
Act authorization (HIPAA) or other privacy authorization prior to any 
participation in study.  
  
Exclusion Criteria 
 
1. Self-reported unstable body weight in the last 3 months (> ±5%);   
2. Significant changes in the quality of the diet or the level of physical activity 
within the last 3 months;  

 
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3. Weight >396 lb (180 kg); 
4. Diagnosis of Type 1 or Type 2 diabetes mellitus;  
5. Fasting blood glucose ≥126 mg/dL;  
6. HbA1c ≥ 6.5%  
7. Blood pressure >140/90 mmHg ;   
8. Self-reported history or presence of the following cardiovascular 
conditions: congestive heart failure, coronary artery disease, significant 
valvular disease, congenital heart disease, serious arrhythmia, stroke, 
symptomatic peripheral artery disease, stable angina, myocardial infarction or 
coronary revascularization within 6 months; clinically significant abnormalities 
on ECG, presence of cardiac pacemaker, or implanted cardiac defibrillator; 
inability to complete the VO2max test; 
9. Anemia (hemoglobin <12 g/dl in men or 11 g/dl in women; Hct <34%); 
10. Kidney disease (creatinine >1.6 mg/dl or estimated glomerular filtration rate 
<60 mL/min/1.73m2); 
11. Abnormal liver enzymes (>2 times the upper limit of normal) that are 
clinically significant;   
12. Serious digestive disorders including severe gastroesophageal reflux 
disease (GERD), irritable bowel syndrome (IBS), Crohn’s disease, chronic 
constipation and/or diarrhea, etc.;   
13. Self-reported chronic, active, or latent infection requiring chronic antibiotic 
or anti-viral treatment; Human Immunodeficiency Virus (HIV); active hepatitis 
B or C undergoing antiviral therapy; 
14. Current or history of eating disorders;   
15. Abnormal bleeding or coagulopathy (self-report) or history of a bleeding 
disorder or clotting abnormality; 
16. Clinical diagnosis of Chronic Obstructive Pulmonary Disease (COPD); 
17. Dyslipidemia (fasting triglycerides >500 mg/dL; low-density lipoprotein 
cholesterol (LDL-C) >190 mg/dL); 
18. Whole blood donation in the last 3 months or platelet/plasma donation in 
the last week or plans for donation during entire protocol period; 
19. Receiving active treatment (including monoclonal antibodies) for 
autoimmune disorders within the last 6 months; 
20. Alcohol consumption >7 drinks per week for women or 14 drinks per week 
for men or history of binge drinking (≥5 drinks for males or 4 drinks for females 
in a 2-hour period more than once per month); 
21. Current smokers (smoking or use of any tobacco or e-cigarette/e-
nicotine/THC/cannabis products in the last 3 months); stopped smoking <10 
years at time of screening for those with a >20 pack-year smoking history or 
stopped smoking <5 years at time of screening for those with a <20 pack-year 
smoking history; 
22. Night shift work in the last 6 months or planning night shift work during the 
study period; 
23. Known allergy to lidocaine or other local anesthetic; 

 
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24. Hyperthyroidism including those with normal TSH (0.35-4.5) on 
pharmacological treatment; individuals with hypothyroidism may be referred 
to their primary care provider for evaluation and retested; any medication 
change for hypothyroidism must be stable for ≥3 months prior to retesting; 
25.  Previous bariatric or other surgery for obesity; 
26. History of cancer (other than non-melanoma skin cancer) within the last 5 
years (not in remission); anti-hormonal therapy (e.g., for breast or prostate 
cancer) within the last 6 months; 
27. Diagnosed psychotic or psychiatric conditions prohibiting adherence to 
study protocol; hospitalization for any psychotic or psychiatric condition within 
one year; 
28. Currently pregnant (pregnancy test performed on day of DEXA scan in 
women of child-bearing potential); post-partum during the last 12 months; 
lactating during the last 12 months; planning to become pregnant during the 
participation period; 
29. Polycystic Ovarian Syndrome (PCOS) (self-report); 
30. Metabolic bone disease (self-report) including history of non-traumatic 
fracture from a standing height or less and/or current pharmacologic treatment 
for low bone mass or osteoporosis, other than calcium, vitamin D, or estrogen; 
31. Hospitalization for COVID-19 infection in the past 12 months; individuals 
who tested positive for COVID-19 but were not hospitalized must be symptom-
free at least 14 days; 
32. Partial and/or full hysterectomy (self-report); 
33. Not willing to archive biospecimens for future use; 
34. Not willing to undergo the microdialysis procedure; 
35. Participants who fulfill any of the contraindications for MRI including 
metal implants, devices, paramagnetic objects contained within the body and 
excessive or metal-containing tattoos; 
36. Unable to participate in MR assessments due to claustrophobia or physical 
limitations of equipment tolerances (e.g., MRI bore size) based on 
investigator’s judgment at screening; 
37. Positive toxicology result from screening;  
38. The following medication use is exclusionary:  
• Dose change for any chronic-use drug in the last 3 
months;  
• Cardiovascular: beta blockers and centrally acting 
anti-hypertensive 
drugs, 
anticoagulants, 
antiarrhythmics, and antiplatelet drugs (other than 
aspirin ≤100 mg/day);  
• Psychiatric: chronic use of medium- or long-acting 
sedatives 
and 
hypnotics, 
including 
all 
benzodiazepines (short-acting non-benzodiazepine 
sedative-hypnotics are allowed), mood stabilizers, 
antiepileptic 
drugs, 
stimulants, 
Attention-

 
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Deficit/Hyperactivity Disorder (ADHD) drugs, anti-
psychotic drugs;  
• Pulmonary/Inflammation: chronic oral steroids 
(inhaled steroids are allowed), burst/taper oral 
steroids more than once in the last 12 months, B2-
agonists (allowed if on stable dose at least 3 months); 
• Genitourinary: 5-alpha reductase inhibitors, daily 
phosphodiesterase type 5 inhibitor;  
• Hormonal: androgenic anabolic steroids, anti-
estrogens, 
anti-androgens, 
estrogens 
and/or 
progestins used for reasons other than birth control, 
growth hormone, insulin like growth factor-I, growth 
hormone releasing hormone, any drugs used to treat 
diabetes mellitus or lower blood glucose, metformin 
for any indication, any drugs used specifically to 
induce weight loss, any drugs used specifically to 
induce muscle growth/hypertrophy or augment 
exercise-induced muscle hypertrophy;  
• Pain/Inflammation: narcotics and narcotic receptor 
agonists, 
regular 
use 
of 
non-steroidal 
anti-
inflammatory drugs (NSAIDs) or acetaminophen, 
muscle relaxants ≥2 days per week;  
• Other: chronic systemic antimicrobials (antibiotic, 
antiviral, antifungal, antiparasite, etc.) for any 
reason, high-potency topical steroids if ≥10% surface 
area using rule of 9s, continuous/chronic use of 
antibiotics or other anti-infectives for treatment or 
prevention, monoclonal antibodies, anti-rejection 
medications/immune suppressants; 
39. Presence of any condition that, in the opinion of the Investigator, 
compromises participant safety or data integrity or the participant’s ability to 
complete the study. 
 
Resources Available 
 
We attest that all AdventHealth Translational Research Institute faculty and staff will be trained 
and this training will be documented as described in AdventHealth Translational Research Institute 
Work Instruction 031.100.015 “Documentation of Protocol Training”. All PBRC investigators and 
staff involved in the project will be appropriately trained to conduct each element of the research 
in accordance with standard operating and quality control procedures, with knowledge of racial, 
ethnic, and cultural appropriateness. In addition, the study team has extensive expertise in topics 
aligned with the project, including nutrition, obesity, physical activity, physiology, adipose tissue, 
energy metabolism, and statistics. All methods and procedures in the project have been 
meticulously authenticated and are well established in the laboratories of AH TRI and PBRC, and 

 
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the analyses will be conducted in compliance with established Good Laboratory Practice 
methodological protocols. 
  
We will implement regular, ongoing discussions between the principal investigator (PI) and 
coordinator as per the AdventHealth Translational Research Institute SOP 030.000.002 “Oversight 
of Research Studies at the Translational Research Institute”. The coordinator will review source 
and communicate with all applicable study team members involved in the study on a regular basis 
regarding reportable new information, implementing amendments, study progress, and quality 
assurance/control.  PBRC will remain in communication with TRI through the duration of the 
study to ensure that all required approvals have been obtained, that each site has the most current 
version of the protocol, consent document, and HIPAA authorization that includes any recent 
modifications, and that all data will be safeguarded as required by local information security 
policies  
  
The AdventHealth Translational Research Institute facilities are state of the art, and we have 
within our building all the required resources and staff to execute the study. We have a medical 
oversight team, Medical Oversight Committee, as well as a Quality Committee to appropriately 
monitor and address adverse events. The multidisciplinary team at PBRC is well suited to 
conduct the study in alignment with the experimental design and to provide appropriate safety, 
oversight, and assessment of adverse events with all procedures (e.g. Medical Investigator and 
nurse practitioners).  
 
Other members of the study team include Dr. Robbie A. Beyl (biostatistician at PBRC) and Dr. 
Marc Hellerstein (consultant). Dr. Beyl’s primary responsibilities for this research will be clinical 
intervention design, creation of the randomization scheme, and conducting the statistical analyses. 
Dr. Hellerstein developed the method of 2H-labeling for quantification of adipocyte formation and 
TG turnover rates in vivo in mice and humans and has a long history of collaborating with Dr. 
White. Dr. Hellerstein will provide support for the data analyses and interpretations. 
 
Study Procedures 
 
Subject Recruitment and Screening 
 
Recruitment methods utilized may include, but will not be limited to, the following: 
recruitment from within the AdventHealth TRI or Pennington Biomedical Research 
Center patient population, electronic medical records and database searches (including 
third party recruitment vendors); advertising in multiple media such as print ads, flyers, 
brochures, posters; radio ads; television spots; and internet/social media advertising.  All 
advertising materials will be submitted to the AdventHealth Institutional Review Board 
(IRB) for review prior to using or publishing them. Recruitment efforts will follow 
AdventHealth Orlando recruitment SOPs for research.   
 
Consent Process  
 

 
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We attest that all study staff delegated the authority to obtain informed consent will 
follow CW AHC 216, “Informed Consent Process and Written Documentation of 
Informed Consent”. Upon consent, a research team member will provide the participant a 
copy of the signed consent and the original consent form placed in the participants chart. 
    
Subjects who are not yet adults (infants, children, teenagers) 
 
• N/A 
 
Cognitively Impaired Adults 
 
• N/A 
 
Adults Unable to Consent 
 
• N/A 
 
Documentation of Informed Consent Process 
 
 
Documentation of the informed consent process is required to establish that the subject 
was accurately and adequately informed and that no study-related procedures were 
initiated prior to obtaining informed consent. A research team member will note in the 
source documentation the consent process, date consent was obtained, and that consent 
was obtained prior to initiating any research procedures.  
 
Waiver of Written Documentation of Consent or Waiver of Consent  
 
Waiver of Written documentation of Consent (consent will be obtained but 
signatures will not be required)   
 
N/A 
 
Waiver or Alteration of the Consent Process (consent will not be obtained, 
required information will not be disclosed, or the research involves deception) 
 
N/A 
 
Waiver or Alteration of HIPAA Authorization 
 
Partial waiver of HIPAA authorization will be requested.  
 
Non-English Speaking Subjects 
 

 
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We intend to enroll only English-speaking participants due to the complexity of the study 
procedures and the lack of translational capacity that could be adequately provided 
throughout the duration of the study. 
 
Randomization 
 
Men and women will be randomized to the CTRL and EX groups on a 1:1 basis 
following successful completion of the VO2max test and imaging tests. Participants will 
be stratified by sex to ensure balance between each of the groups. Stratifying allows for 
different sample sizes in each sex, but ensures that within each sex, there are an equal 
number of treatments and controls.  
  
Study Visits  
 
Study visits will be conducted at the AdventHealth Translational Research Institute or 
Pennington Biomedical Research Center.   
  
Screening Visit (SV) (Outpatient; Approximate visit time: 2 hours)   
 
Participants will arrive for this outpatient visit after a 10 hour overnight fast. After 
obtaining informed consent, data collection for assessing eligibility will commence.  The 
screening visit will include:  
  
1Urine for pregnancy test (if applicable), urinalysis, and toxicology screen 
(Amphetamine, Barbiturate, Benzodiazepine, Cocaine, Methadone, Opiate, THC, 
Cannabis, Tricyclics, and Oxycodone)  
 
2Demographics and complete medical history (including, but not limited to, alcohol use, 
concomitant medications, health conditions, allergies and exercise habits)   
 
3Vital signs (respiratory rate, temperature, heart rate, blood pressure)  
 
4Standard physical examination performed by a study physician, physician assistant, or 
nurse practitioner 
 
5Anthropometrics including height, weight, waist circumference, and BMI  
 
6Screening blood draw to measure CBC with differential and platelets, comprehensive 
metabolic panel (CMP), lipid profile, blood glucose, HbA1C, ALT, AST, alkaline 
phosphatase, insulin level, and TSH 
 
7Electrocardiogram (ECG)  
 
8Consent Process Checklist assessing the potential participants’ understanding of and 
willingness to perform all study activities. 

 
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Upon review of all clinical and laboratory-based screening endpoints, eligible 
participants will then be scheduled for baseline assessments. Laboratories can be repeated 
and participants may be rescreened for the study at the PI or provider’s discretion.  
  
Baseline Period – Visit 1 (V1)   
  
Day -14 (±2): Outpatient. Approximate visit time at TRI: ~ 3-4 hours  
  
1Anthropometrics including height, weight, waist circumference, and BMI.  
 
2Vital signs including respiratory rate, temperature, heart rate, and blood pressure 
  
3MR assessment of visceral and subcutaneous fat (VAT/SAT) and thigh fat (~1 
hour)*: Volumetric measurement of fat, muscle and bone in the participant’s abdomen and 
thigh will be completed using an Achieva 3T MRI/Multinuclear MRS (Philips, Andover 
MA), a Siemens 3T Prisma (Siemens Healthcare, Erlangen, Germany) or a GE Discovery 
750w.  Scans will be performed under standardized conditions with subjects in a supine 
position using the quadrature body coil.  Each scan will take approximately 45 minutes to 
perform. To minimize the potential for confounding factors to affect the measurements, 
MR scans will be conducted following a fast (except water) of at least 4 hours. Low 
resolution scans will be taken to determine appropriate positioning for high resolution 
images.  Then, T1 weighted imaging sequences will be performed covering the entire 
abdomen, ensuring complete coverage from the diaphragm through the pubic 
symphysis.  Participant will be repositioned supine feet first, to scan similar images over 
the thighs.   
 
Fat Quantification:  Resultant images will be analyzed using Analyze (Biomedical Imaging 
Resource, Mayo Clinic, Rochester MN) to segment depots of fat (eg. subcutaneous vs. 
visceral) as well as bone and muscle volume. 
 
Incidental Findings: There will be no diagnostic analysis associated with any of the MR 
sequences used in this protocol.  However, some of the MR images we obtain as part of 
this protocol may show incidental medical findings.  In the case where a medical 
abnormality is apparent on an image, the image will first be reviewed by an investigator on 
this protocol.  If the abnormality is confirmed, then the participant will be instructed to 
seek medical attention from their health care provider.  
 
4Dual Energy X-Ray Absorptiometry (DEXA)*: DEXA Scans will be performed to 
measure body fat and estimate muscle mass using a GE Lunar iDXA whole-body 
scanner.  The participant will remove all metal accessories and may be asked to change 
into a hospital gown.  The participant will lie on the DEXA table while the scanner arm 
emits low energy X-rays as it passes along the body.  The scan takes up to 15 minutes and 
the radiation dose is less than 1 mrem, less than half the average daily radiation dose in 
America.  A urine pregnancy test will be completed on all women of childbearing potential 

 
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(all women except those with prior hysterectomy, tubal ligation, or absence of menses for 
>2 years) prior to the DEXA scan for safety.  
 
5Maximal Oxygen Consumption: Aerobic fitness will be determined by measuring 
maximal volume of oxygen consumed (VO2max) during a stationary bicycle exercise test. 
Provider monitoring will follow The American College of Sports Medicine (ACSM) 
Guidelines for Exercise Testing and Prescription.  During the test, participants will breathe 
through a low resistance mouthpiece and wear a nose clip. Expired gases will be measured 
by indirect calorimetry and heart rate/rhythm monitored by 12-lead EKG.  Participants will 
perform an exercise test on a stationary bicycle ergometer (Lode Excaliber, Gronigen, The 
Netherlands) at the local site. The test will begin with a warm-up of light pedaling. After 
the 2-minute warm up at a constant workload, the workload will increase in a ramped 
manner that is individualized to the participant (either 10watts/minute or 15watts/min) 
based on participant characteristics. VO2max tests will take as little time as possible in order 
to ensure that test termination is due to the subject reaching VO2 max rather than terminating 
the test due to muscle exhaustion. There are several additional criteria that will be used to 
determine if maximal aerobic capacity is achieved. These criteria are the respiratory 
exchange ratio (RER) increased to 1.05 or higher and the subject’s heart rate increased to 
within 10 beats of the age-predicted maximum (208 – [0.7 x age]). This test will be 
supervised by a qualified Medical Provider as stated in “TRI SOP 030.100.015 Procedural 
Staffing.”. All study investigators and staff involved in the project are appropriately trained 
to conduct each element of the research in accordance with standard operating and quality 
control procedures. 
  
6Randomization: Once a participant passes screening, completes the VO2max test, and is 
considered eligible to continue in the study, they will be randomized in a 1:1 ratio into 
either the control (CTRL) group or the exercise group (EX). Randomization will be 
conducted by a statistician.  
  
Baseline Period – Visit 2 (V2)   
  
Day-8 (± 2): In patient. Participants will report to the local site the evening of day -8 for 
an overnight stay. A single urine collection will be done for measurements of urea, 
nitrogen, and creatinine. Participants will then receive a standardized dinner meal (10 
kcal/kg; 50% carbohydrate, 15% protein, 35% fat), and be strongly encouraged to 
consume all food provided. Any unconsumed food will be recorded. Participants will 
remain fasting from completion of evening dinner meal at the local site until WAT biopsy 
and hyperinsulinemic-euglycemic clamp protocol are completed the following day.  
 
1Surveys (Optional): 
 
With the exception of the Diet History Questionnaire (DHQ) III, all surveys will be 
administered to participants via the password protected computer-based software 
REDCap.  
 

 
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• Diet History Questionnaire (DHQ) III: Approximate duration: 10 min; the DHQ-
III, owned by the NIH National Cancer Institute will be completed through the NIH-
developed secured platform specific for this instrument. Participants will complete 
the DHQ-III under their assigned PID during the clinic visit and no personal 
identifiers will be obtained. 
• Retrospective Visual Analog Scale (VAS): Approximate duration: 5 min; an 8-item 
retrospective VAS assessing appetite, including hunger, fullness after meals, thoughts 
of food, cravings, and urges to eat. 
• Three Factor Eating Questionnaire (TFEQ): Approximate duration: 5 min; an 18-
item questionnaire assessing cognitive and behavioral components of eating and 
comprises 3 different scales corresponding to cognitive restraint, emotional eating, 
and uncontrolled eating. 
• Food-Craving Inventory (FCI): Approximate duration: 5 min; a 33-item self-report 
measure of general and specific food cravings relating to overeating and binge eating. 
• MacArthur Socioeconomic Status Index: Approximate duration: 6 min; an 11-item 
questionnaire regarding participants’ perception of their socioeconomic status relative 
to their community and country, their living situation, level and degree of education 
obtained, job status, income, and debt. 
• Perceived Stress Scale (PSS): Approximate duration: 3 min; a 10-item questionnaire 
measuring the degree of perceived stress regarding certain life events during the past 
month. 
• Generalized Anxiety Disorder 7-item (GAD-7) scale: Approximate duration: 3 
min; a 7-item brief measure for symptoms of anxiety, based on the generalized 
anxiety disorder diagnostic criteria described in the Diagnostic and Statistical Manual 
of Mental Disorders (DSM). 
• Center for Epidemiologic Studies Depression Scale (CES-D): Approximate 
duration: 3 min; a 20-item measure for symptoms associated with depression, such as 
restless sleep, poor appetite, and feeling lonely. If a participant’s score is ≥16, the 
REDCap survey platform will trigger an alert to the study coordinator. A study 
provider will meet with the participant and direct them to resources/referrals as 
needed based on provider assessment and discretion. 
 
2Hair Collection (Optional): A hair sample will be cut with fine scissors from the 
posterior vertex position of the scalp and as close to the scalp as possible. Approximately 
40 hair follicles will be cut (slightly larger than the diameter of a pencil) to reach the 
needed weight of 50 mg. The sample will be stored for future analyses. 
  
Day-7: Early morning.  
  
3WAT Biopsies: The abdominal skin 6-10 cm lateral to the umbilicus will be cleansed 
with chlorhexidine. A sterile drape will be placed, and the skin and adipose tissue will be 
anesthetized using 1%- Lidocaine HCl and a 0.09% Tumescent Lidocaine solution. A 3-4 
mm Mercedes Liposuction needle will be inserted for aspiration of a minimum of 1.5g up 
to 10g of adipose tissue. Once the sample is aspirated, the needle will be removed. In the 
instance where the use of a needle under aspiration yields inadequate amounts of adipose 

 
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tissue, additional adipose tissue may be collected using forceps and a scalpel to excise 
visible superficially located subcutaneous adipose tissue. The incision will be closed with 
steri-strips (suture(s) if allergy to steri-strips), and sterile dressing will be applied. An 
MD, DO, NP, or PA will conduct this procedure. The adipose tissue will be processed as 
follows:  
 
1. Fresh procedures and storage  
a. 
Adipose tissue cell isolation and flash frozen for isotope labeled 
DNA extraction.  
2. Histology (Formalin fixed)  
a. 
Fat cell size   
b. 
Other  
3. Frozen  
a. 
snRNAseq  
b. 
TG turnover quantification  
  
4Muscle Tissue Biopsies (Optional): A biopsy of the Vastus Lateralis muscle will be 
performed using the Bergstrom technique.  Subjects will be placed in a supine position and 
the skin will be cleansed with chlorhexidine solution.  After a sterile drape is placed over 
the skin, local anesthesia (1%-Lidocaine HCl) will be administered,. The skin will be 
incised (approximately 0.75 cm) with a #11 scalpel, and the Bergstrom needle will be 
inserted into the Vastus Lateralis.  Up to 250mg of muscle tissue will be obtained under 
suction. After the biopsy, pressure will be applied to stop bleeding and the skin will be 
closed with Steri strips (suture(s) if allergy to steri strips).  A sterile dressing will be 
applied.  An MD, DO, NP, or PA will conduct this procedure. Collected tissue will be snap-
frozen for future analyses.   
  
5Hyperinsulinemic-euglycemic Clamp: Hyperinsulinemic-euglycemic clamp: The 
glucose clamp is the gold standard for measurement of insulin sensitivity and will be 
performed similarly to the procedure initially described by Defronzo in 1979. The overall 
concept of the euglycemic clamp is that insulin is infused to stimulate glucose uptake and 
inhibit endogenous glucose production, and the rate of 20% glucose infusion necessary to 
maintain euglycemia can be used to assess whole body insulin sensitivity.  
  
After an overnight 10-hour fast, an intravenous catheter will be placed for infusion of 
insulin and glucose.  A second catheter will be placed in a vein of the contra-lateral arm 
for blood withdrawal. After baseline blood is collected, a 2-step euglycemic clamp will be 
started with a 2-hour infusion of insulin (Humulin-R) at 10 mU/m2/min, followed by 2 
hours at 40 mU /m2/min.  Whole blood will be used to assess plasma glucose at 5-10 min 
intervals via NOVA StatStrip Glucose Meter or YSI. Plasma glucose will be allowed to 
either decrease to ~90 mg/dl or will be increased to ~90 mg/dl in each participant depending 
on the fasting blood glucose levels. Euglycemia (~90 mg/dL) will be maintained by 
measuring blood glucose at 5-10 min intervals via a NOVA glucometer or YSI and using 
a variable 20% dextrose infusion.  

 
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Throughout the clamp blood samples are collected at -30min, -20min, -10min, 0min, 
+45min, +90min, +100min, +110min, +120min, +180min, +220min, +230min, and 
+240min of insulin infusion. Total blood volume during this procedure will be 
approximately 260 mls. 
 
Calculations:  
Insulin sensitivity (M/I) will be calculated using the formula: M/I = Rd / (steady state 
insulin level - basal insulin level) where steady state insulin equals the average insulin 
concentration during the last 30-40 minutes of each steady state phase of the clamp.  
Fasting and insulin-suppressed FFA [IS-FFA] will be measured as a measure of adipose 
tissue insulin sensitivity with an ultrasensitive FFA assay from Wako.  
 
6Subcutaneous Microdialysis: To assess antilipolytic sensitivity of WAT to insulin and 
production of hormones, proteins, and other factors (including, but not limited to amyloid 
precursor protein (APP) TNF-a, leptin, and adiponectin), up to four microdialysis catheters 
will be inserted into the periumbilical subcutaneous adipose tissue. Topical anesthesia 
(EMLA cream) or ethyl chloride will be applied to each insertion site.   
  
The catheters come as a sterilized kit (CMA Microdialysis) with a sterilized cutting needle 
(20-gauge) and introducer needle (54mm long X 1.4 mm diameter).  The insertion site will 
be cleaned with chlorhexidine and a small incision made using the provided cutting 
needle.  The catheter will be inserted using the introducer needle, which is then removed, 
and the catheter secured under a clear adhesive dressing.  Flow to the microdialysis 
catheters will be initiated and dialysate collected in approximately 30-minute samples; the 
catheters will be calibrated using zero-net flow methods.  Perfusate will consist of sterile 
10 mM ethanol in ringers injection to account for changes in adipose tissue blood flow. The 
microdialysis collection will proceed during the glucose clamp. Approximately 66 mL of 
blood and less than 8 mL of interstitial microdialysate will be obtained.    
 
The rates of carbohydrate and lipid oxidation will be calculated as described by Elia and 
Livesey.  The basal rate will be calculated by averaging the data for the time= -20 to time=0 
minutes (fasting RQ), and insulin-stimulated glucose and lipid oxidation rates will be 
calculated by averaging the data for the time=40 to time=60 minutes (post glucose 
administration RQ). 
 
7Blood Draw: After the IV catheters are inserted and prior to the clamp procedures 
beginning, a baseline blood draw will be collected from the venous catheter. The following 
will be measured: CBC with differential and platelets, CMP, lipid profile, blood glucose, 
ALT, AST, alkaline phosphatase, insulin level, TSH, and HbA1C. 
 
8Respiratory Quotient (RQ): Indirect calorimetry will measure the respiratory quotient 
(RQ) and substrate utilization using either a ParvoMedics Metabolic Cart at TRI (Salt Lake 
City, UT) or Omnical Cart at PBRC (Maastricht Instruments BV).  The ParvoMedics 
Metabolic Carts are calibrated daily before each subject with standardized gases containing 

 
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1% CO2, 16% O2 and balance N2. The Omnical Carts are also calibrated daily before each 
subject with standardized gases containing 0.8% CO2, 18% O2 and balance N2 and then 
self-calibrate continuously through the test approximately every 15 minutes. After 
inserting the clamp catheters, completing the WAT and muscle biopsies, and inserting the 
microdialysis catheters, and allowing flushing of the microdialysis catheters for 5 minutes, 
participants will rest for 60 minutes laying down in a Semi-Fowler’s position. After 30 
minutes of dialysate collection, a transparent plastic hood connected to the device will be 
placed over the head of the subject. Calculations of O2 consumption (VO2) and CO2 
production (VCO2) will be made by comparing measures of the O2 and CO2 concentrations 
of the testing room (measured every 5 minutes at TRI and every 2 minutes at PBRC) to 
continuous measurements of O2 and CO2 concentrations in expired air diluted in a constant 
air flow (15-30 L/min at TRI and 85-90 L/min at PBRC) generated by a pump.  From the 
above, energy expenditure standardized for temperature, pressure, and moisture will be 
calculated at one-minute intervals. After ~30 minutes, the remaining clamp procedures will 
resume. This will be repeated during steady state 1 (+90-120min) and steady state 2 (+210-
240mins). 
 
 
 
9Standardized Meal and Accelerometer: After all procedures are complete, participants 
will be provided a standardized meal along with post procedure instructions. Participants 
will also be provided with a triaxial activity monitor (Actigraph GT3X) which will be worn 
for 7-10 days. The monitor integrates motion sensor data from the tri-axial accelerometer 
to estimate the energy cost of free-living activity. The participant will wear the monitor at 

 
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all times, except while showering or bathing. After the period of wear time, the participant 
will return the monitor and the data will be uploaded. 
 
107-Day Food Record (optional): Participants will be asked to record their food and 
beverage intake for 7 days and return the form to the research site at their next in-person 
visit. 
  
Phone Follow-up After Baseline Visit 2  
  
The participant will be contacted by phone within 24-72 hours after baseline visit 2 
discharge to ensure no adverse events have occurred on both biopsy and arterial line sites.  
  
Intervention Period (Weeks 1-12)  
  
Biopsy and catheter insertion site check:  A biopsy site and catheter site check will be 
done when the participants pick up their first supply of 2H2O.  
  
H2O Administration and Return (12-week labelling period) and Urine 
Collections:  2H2O will be prepared by the Mass Spectrometry Core. Participants will 
receive 50 mL aliquots of  70% 2H2O or 35 mL aliquots of 99.9% 2H2O in  individual amber 
prescription containers. Participants will drink three 35 mL or 50 mL doses per day for the 
first week, and two doses per day from the second through the twelfth week. Participants 
will receive the 2H2O aliquots containers bi-weekly, a log sheet to record consumption, and 
at-home urine collection cups with instructions. Participants will be instructed to keep urine 
samples taken at home in the refrigerator until their next in-person visit (every other week). 
Urine samples will be used to verify compliance weekly (at AH TRI or PBRC). 
Compliance with 2H2O intake for both groups will be monitored through the weekly urine 
collections and return of empty containers for counting. 2H enrichments in urine, reflecting 
2H body water enrichments, will be measured by isotope ratio mass spectrometry. 
Enrichments are expected to be between 1-2.5%. If a participant’s urine sample is <1%, 
the study team will encourage compliance with the participant. If enrichment is <1% for a 
period of 3 weeks, the PIs will discuss participant withdrawal. Participants will be 
instructed to maintain their normal daily and weekly routines.  
  
Participants in the EX group will engage in 12-week endurance exercise training.   
  
Endurance Exercise Training: Outpatient. Approximate visit time: ~ 1.5 hour  
Participants in the EX group will engage in endurance exercise training three times a 
week for approximately 12 weeks. The exercise program will be supervised by a certified 
exercise physiologist and will take place at the TRI exercise training facility or at PBRC. 
Each session will last approximately 75 minutes with a 50-60 minute stimulus phase and 
the remaining time being used to warm up and cool down. Participants will use a cycle 
ergometer and/or treadmill for the duration of the intervention period. During all sessions, 
heart rate will be monitored using Zephyr heart rate monitors to ensure that participants 
maintain the target exercise intensity during training. Intensity will be set as a percentage 

 
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of heart rate reserve (calculated from pre-training VO2max) and will increase during the 
intervention: 60% ± 5 bpm for weeks 1-4; 70% ± 5 bpm for weeks 5-8; 75% ± 5 bpm for 
weeks 9-10; and 80% ± 5 bpm for weeks 11-12. Self-reported perceptual data will be 
recorded periodically during exercise sessions and will be used to track the subjective 
experience of participants and in interpreting adherence data.   
  
Body Weight Management and Health Education: All participants will be expected to 
maintain weight within ±2kg throughout the study. Should a participant gain or lose weight 
>2kg, the study team will be instructed on how to reduce or increase caloric intake to 
maintain body weight (Appendix). Participants in the EX group will be weighed onsite 
weekly and the study team will advise on weight maintenance, if needed. Participants in 
the CTRL group will be weighed onsite biweekly when they return and pick up 2H2O. 
During biweekly on-site weigh ins, CTRL participants will be instructed on weight 
maintenance, if needed. Additionally, the study team will review the participant’s physical 
activity habits and dietary habits, adverse events, and medication/medical history changes.  
 
DEXA (Weeks 4-6): In addition to the pre- and post-intervention MRI and DEXA scans, 
during Weeks 4-6, one DEXA scan will be performed as described above  to confirm 
maintenance of fat mass.  
  
Review of Adverse Events: Study staff will assess for any adverse events at each exercise 
visit.  
 
Accelerometers: Following the last bout of exercise training, participants will be provided 
with a triaxial activity monitor (Actigraph GT3X) which will be worn for 7-10 days. The 
monitor integrates motion sensor data from the tri-axial accelerometer to estimate the 
energy cost of free-living activity. The participant will wear the monitor at all times, except 
while showering or bathing. After the period of wear time, the participant will return the 
monitor and the data will be uploaded. 
 
7-Day Food Record: Participants will be asked to record their food intake for 7 days 
during Week 12 and return the form at Visit 3.  
  
Follow-up Period – Visit 3  
  
Day 85 (±1): Outpatient. Approximate visit time: ~3-4 hours. Scheduled 48-72 hours 
after the last exercise bout. 
 
The following assessments will be performed as described in Baseline Period – Visit 1: 
   
1Anthropometrics including height, weight, waist circumference, and BMI.  
 
2Vital signs (respiratory rate, temperature, heart rate, blood pressure)  
 
3MR assessment of visceral and subcutaneous fat (VAT/SAT) and thigh fat (~1 hour)  
 

 
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4Dual Energy X-Ray Absorptiometry (DEXA) 
 
5Maximal Oxygen Consumption 
 
Follow up Period – Visit 4  
  
The following assessments will be performed as described in Baseline Period – Visit 2: 
 
Day 87 (± 2): In patient. Participants will report to the local site the evening of day 87 for 
an overnight stay. Urine collection will also be done for measurements of urea, nitrogen, 
and creatinine. Participants will then receive a standardized dinner meal (10 kcal/kg; 50% 
carbohydrate, 15% protein, 35% fat), and be strongly encouraged to consume all food 
provided. Any unconsumed food will be recorded. Participants will remain fasting from 
completion of evening dinner meal at the local site until WAT biopsy and 
hyperinsulinemic-euglycemic clamp protocol are completed the following day.   
 
1Surveys (Optional)  
• Diet History Questionnaire (DHQ) III 
• Retrospective Visual Analog Scales (VAS) 
• Three Factor Eating Questionnaire (TFEQ) 
• Food-Craving Inventory (FCI) 
• Perceived Stress Scale (PSS) 
• MacArthur Socioeconomic Status Index 
• Generalized Anxiety Disorder 7-item (GAD-7) scale 
• Center for Epidemiological Studies-Depression Scale (CES-D) 
 
2Hair Collection (Optional) 
  
Day 88 (± 2): Early morning.  
 
3WAT Biopsies 
  
4Muscle Tissue Biopsies (Optional) 
  
5Hyperinsulinemic-euglycemic Clamp  
 
6Subcutaneous Microdialysis 
 
7Blood Draw 
 
8Respiratory Quotient (RQ) 
  
Phone Follow-Up After Visit 4 
  

 
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The participant will be contacted by phone within 24-72 hours after Visit 4 discharge to 
ensure no adverse events have occurred on both biopsy and arterial line sites.  
   
A table identifying the procedures in relation to the study timeline can be found at the end 
of this document.  
 
 
Study Duration 
 
Projected start date: August 2024   
  
Estimated duration to enroll all study subjects: 4 years  
  
Total length of time participants will remain in the study: approximately 14 weeks  
  
Estimated date for investigators to complete the study (including analysis): April 2029 (5 
years).  
 
Materials of Human Origin:  Collection, Preparation, Handling and Shipping 
 
Materials of human origin will be collected in the manner described in the specific study 
visits section of this protocol.    
  
Biospecimen samples will be stored in ultralow temperature freezers and liquid nitrogen 
drawers, or other storage units located at each facility. The TRI facility is secured via key 
card and equipped with a back-up generator system. Laboratory personnel in the facility 
have 24/7 key-controlled access to the laboratory.  Chain of custody of biospecimen 
samples is maintained through requisition forms and in the StarLIMS database. Specimen 
tubes are coded, and specimen requests and distribution are documented. The PBRC 
facility is secured via key card and each building is equipped with a back-up generator 
system. Laboratory personnel within each core of the facility have 24/7 key-controlled 
access to the respective core laboratory with freezer storage spaces. The chain of custody 
of the biospecimen is maintained through requisition forms that are specific to each core 
and on the PBRC OneDrive. All biospecimen tubes are coded and labeled appropriately, 
and biospecimen requests and distribution are documented. 
  
The TRI facility will be the main repository for biospecimen samples. PBRC will ship all 
tissue and blood samples (with exception of WAT samples needed for triglyceride turnover 
and adipocyte formation analysis and screening/safety visits) to AH TRI for testing and 
storage. The AH TRI will ship WAT samples needed for triglyceride turnover and 
adipocyte formation analysis. Material Transfer Agreements will be in place prior to 
transferring any samples between study sites. The MTA will govern the transfer and chain 
of custody of biospecimens outside of AdventHealth.   
 

 
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The adipose tissue specimens and plasma/serum samples will be stored indefinitely, or 
until a sample is fully used. All biological specimens will be stored without identifiers or 
linkage codes.   
  
After study aims have been achieved and study related endpoints have been measured and 
analyzed, any remaining biospecimens will be stored at the TRI Laboratory Room 2404 
and will then be considered as “archived biospecimens.”  Archived biospecimens will be 
used for any additional hypothesis-related experimentation or testing for the purposes of 
this study, consistent with the original aims, which cannot be predicted at the time the 
protocol is developed due to the evolving nature of scientific exploration.    
  
Additionally, archived biospecimen samples may be stored indefinitely for future 
research. Archived biospecimens could be used for separate research by both 
AdventHealth and PBRC scientists and scientists outside of AdventHealth and PBRC. 
This would be allowed for research of any type (without limitation to disease, process, 
or research methods) if it has scientific merit as determined by the PI, with an additional 
review by the respective Program Director.  For research outside of AdventHealth or 
PBRC, a Material Transfer Agreement will be obtained, which will govern the transfer 
and chain of custody of the biospecimens outside of AdventHealth or PBRC.  
 
Study Outcome Measures (Endpoints) 
 
Primary outcomes:  
1. In vivo adipogenesis in WAT. New adipocyte formation will be assessed two times 
(pre and post intervention) on the frozen isolated 2H2O labelled adipose tissue cells.    
2. In vivo TG turnover. WAT TG turnover rates will be assessed two times (pre and 
post intervention) on abdominal subcutaneous WAT on the extracted lipid from the 
frozen 2H2O labelled adipose tissue.  
3. Cellular composition of WAT. Cell composition and gene expressions in WAT will 
be assessed two times (pre and post exercise intervention) by snRNAseq on the frozen 
adipose tissue.  
4. Adipocyte morphology, senescence, and inflammation. Adipocyte size, markers of 
cellular senescence and inflammation will be assessed two times (pre and post exercise 
intervention) by immunohistochemistry on the formalin-fixed paraffin-embedded 
adipose tissue.   
  
Secondary outcomes:  
1. Whole body and WAT-specific insulin resistance: Whole-body insulin resistance 
and WAT-specific antilipolytic sensitivity to insulin will be evaluated pre and post 
exercise intervention in vivo using blood and dialysate samples collected during 
glucose clamp and subcutaneous microdialysis.   
 
Data Management and Quality Plan 
 

 
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Data De-identification 
 
Participants will be enrolled and assigned a participant identification number (PID). This 
PID is a code consisting of a combination of numerals and letters, which serves as the 
identifier for this participant for this research study and links them back to their hospital 
medical record and their protected health information (PHI). Access to the “link” 
between the PIDs, the PHI, and to the clinical data are only granted to the clinical 
research team as assigned on the Delegation of Authority Log. All the clinical research 
and survey data is recorded in a de-identified fashion onto our paper source documents 
and/or electronic case report forms (CRF). Both sites will enter data into REDCap, a 
HIPAA-compliant, web-based forms and survey platform approved for use by 
AdventHealth. REDCap will be used for storage and to facilitate analysis. All data not 
entered into REDCap (e.g., data collected from individual exercise sessions) will be 
stored on a shared OneDrive folder only accessible to the assigned study-specific 
research team at PBRC and AH TRI. 
 
Clinical data generated by research devices (e.g., accelerometer) also uses the PID, and 
once the data has been transformed into interpretable results it is stored into the clinical 
research database (either REDCap or OneDrive). Both storage locations are password-
protected, secured and only assessable to the assigned clinical research team from AH 
TRI and PBRC. The “link” will not be used to re-identify participants except in the event 
of a serious adverse event (SAE) requiring “unblinding” to treat the participant. The 
“link” will be stored in the Patient Protocol Manager and in the OneDrive folder, where 
only the research team has access.   
     
Data Confidentiality, Storage, and Retention 
 
The identity and personal health information of study participants will be kept confidential 
to the extent permitted by the applicable laws and/or regulations and will not be made 
publicly available. If results of this study are published or presented, the identities will not 
be revealed. Confidentiality will be maintained during and after the study. This information 
is also included in the informed consent, which is discussed with participant prior to 
enrollment.  
  
Study documentation and paperwork will be stored in our locked medical records room at 
AH TRI and PBRC. The data records will also be stored as electronic records in REDCap 
and OneDrive. This data will be safeguarded so that only those on the research team have 
access to any of the clinical data (both source documentation and data warehouse storage). 
The electronic data is maintained by Adventist Information Technology (AIT) security 
controls.    
  
AdventHealth Translational Research Institute retention policy is maintained in the 
Records Management Policy. Electronic de-identified data will be kept indefinitely in our 
data warehouse.   
  

 
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Per the institutional policy, investigator records must be kept for a minimum of 7 years 
after completion of discontinuation of the study, or for longer if required by applicable 
local regulations, or what is applicable per the Sponsor. 
 
Data Quality  
 
Data quality control will occur according to our SOPs on Data Entry, Quality Control 
Procedures and Query Management. All data will be entered into an electronic data 
capture (EDC) system (REDCap) and checked against the paper source for accuracy by a 
second party (Data Entry SOP) and errors resolved through the Query Management SOP.  
Ten percent of the data points will be routinely checked at the beginning, middle, and 
close of a study for quality control (Quality Control SOP).  Finally, all critical endpoints 
(as determined by the PI or Sub-I) will be assessed using quality control analyses.  The 
data will be loaded into the clinical research database.  Data in the warehouse will also be 
routinely monitored over time.  
 
Data Sharing  
 
Some of the endpoint testing will be conducted at outside laboratories/institutions. To 
perform these analyses/testing/etc. and to interpret results, certain data elements will need 
to be shared along with the biospecimen samples. Data Use Agreements will be obtained, 
which will identify the specific data elements to be shared and will govern the sharing of 
data related to this study. Data will be de-identified, but a link/code is managed within an 
electronic research management system and maintained by a study coordinator.   
  
Sample Size Determination 
 
The primary aim is powered to detect differences in the change in in vivo formation of new 
adipocytes (i.e., fraction of newly formed adipocytes; “f”) of subcutaneous WAT between 
the CTRL and EX groups. We hypothesize that the EX-group will have a decrease in the 
fraction of newly formed adipocytes in response to the EX intervention as compared to the 
CTRL group. Analysis of women from our previous VIVA study (NCT03530111) 
indicates that the mean value of “f” in the physically active (exercise) group is 11.4%, 
while the sedentary group has an “f” value of 17.8%. Because VIVA only has cross-
sectional data (assessment at one time-point), we must also use data from the Apple & Pear 
study (NCT01748994) 9 to estimate the change in fraction of newly formed adipocytes, as 
the proposed study includes an exercise intervention. By utilizing data from the CTRL-
group in the Apple & Pear study 9 (baseline assessment) and increasing the estimate to 
ensure that the study is well-powered, we can estimate the CTRL and EX groups from the 
proposed study to have an overall mean “f” value of 17% (at baseline) with a standard 
deviation of 6.0%. A total sample size of 58 (assuming a ~17% overall lost-to-follow-up 
rate) will provide at least 80% power to detect significant differences in “f” between the 
CTRL and EX groups, with a 5% significance level.   
 

 
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Single nuclei RNAseq Power Calculation. To date the most comprehensive analysis of 
human WAT has been conducted by Emont and colleagues who performed snRNAseq in 
whole frozen WAT and VAT from humans with obesity and established 17 subpopulations 
of cells. However, these data were generated using the 10X Genomics platform. We have 
published preliminary snRNAseq data in WAT from 22 individuals with overweight and 
obesity. We expect to capture nuclei from the WAT that may reveal up to 12 different cell 
populations (Fig. 4). If we assume the rarest cell type is 2% and require at least a 100 of 
the of rarest cell type to accurately quantify and describe it, we require a total of 6,792 
nuclei to achieve a 99% probability of meeting these requirements. Our current technique 
permits 1400 cells per participant to be analyzed which equates to 28,000 cells for 20 
participants at each timepoint in EX-group and 14,000 nuclei for 10 participants at each 
timepoint in the CTRL-group. This would allow us to accurately define subpopulations 
within WAT and generate sufficient data to compare the impact of endurance exercise on 
adipocyte populations and non-adipocyte populations.   
 
Statistical Analysis Plan 
 
Primary and Secondary Objective Analysis  
 
Shapiro-Wilk test will be initially performed to assess the normality of data and 
homogeneity of variances. Pre- and post-intervention results will then be tested by paired 
t-test or Wilcoxon signed-rank test, as appropriate. Results will be expressed as mean ± 
standard deviation and significance will be established, a priori, at p≤0.05.  
 
Potential Risks and Benefits 
 
Potential Benefits 
 
The potential benefits to an individual participant in this study are not known. Exercise 
training can trigger many beneficial adaptations, but individual responses and adaptations 
vary. All participants will acquire knowledge about their physiological status (e.g., 
cardiorespiratory fitness, body composition, etc.) that may be of personal benefit.   
The risks presented to participants are minimal, and information gained from this project 
will be useful to identify mechanisms responsible for exercise effects on adipose tissue 
quality and insulin resistance in subjects with obesity. 
 
Potential Risks 
 
 
Described below are all the known risks of being in this study.  
  
Dual Energy X-ray Absorptiometry (DEXA): There is a very small risk of cancer with 
excessive exposure to any radiation.  There are also risks for unborn children associated 
with radiation exposure. The radiation dose from the scan is less than a chest x-ray, or less 
than half the average amount a person would receive in a day in America. 

 
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Magnetic Resonance Imaging: There are no known biological risks associated with 
magnetic resonance imaging. Some short-term discomfort may be experienced.  The short-
term risks associated with MRI are minimal, but include heating, loud noises and 
claustrophobia.  There are some people who should not undergo MRI; the contraindication 
is largely based on the presence of metal objects within a person (i.e. pacemaker, aneurysm 
clip, metal fragments, etc.).    
  
Maximal Oxygen Consumption (VO2max) test and exercise training: There is a risk of 
changes to blood pressure, irregular, fast or slow heart rhythm, fainting, and in rare 
instances (<0.01%) heart attack, stroke, or death with exercise testing, at similar rates to 
exercising during daily life.    
 
Hair Collection: There is minimal risk associated with obtaining hair samples. A rare risk 
includes minor scalp laceration during sample collection and related infection. 
  
Adipose Tissue Biopsy: There is a risk of pain from the local anesthesia, and a risk of 
bruising, bleeding and infection at the site of the biopsy. In addition, there is a risk of skin 
irritation due to the steri strips and dressing.   
  
Muscle Tissue Biopsy: There is a risk of pain from the local anesthesia, and a risk of 
bruising, bleeding (hematoma) and infection at the site of the biopsy. In addition, there is 
a risk of skin irritation due to the steri strips and dressing. Local sensory loss may occur by 
cutting a subcutaneous sensory nerve (<1 in 100), which is almost always temporary but 
occasionally can become permanent. On rare occasion (<1 in 1000) bleeding, requiring 
hospitalization, may occur. The procedures are well tolerated when performed properly.  
  
Intravenous lines/blood draws (lab samples, e.g.): there is a risk of pain, vasovagal 
syncope, hematomas, and/or infection at IV insertion/blood draw site (low risk of SAE).    
  
Subcutaneous Microdialysis: There is a risk of pain, hematomas, and/or infection at 
insertion site (low risk of serious AEs).   
  
Deuterium-labeled water (2H2O) administration: 2H2O-labeled water is water that is 
labeled with the stable, non-radioactive isotope deuterium. Deuterium-labeled water poses 
minimal health risk associated with drinking it. Participants may rarely experience transient 
dizziness or nausea following the initial intake.  
  
Hyperinsulinemic-euglycemic clamp: There is the risk of bruising at the site of needle 
insertion in the veins. The risk of receiving an insulin infusion is that it could produce 
hypoglycemia (rare). Hypoglycemia may cause one to feel nervous, shaky, sweaty, hungry, 
and dizzy and have a rapid heart rate.  If severe, hypoglycemia can cause coma, seizure or 
even death (all rare).   
 

 
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Resting Metabolic Rate/Respiratory Quotient (RMR/RQ): There is no physical risk 
associated with RMR/RQ.  Other risks include a feeling of claustrophobia experienced by 
some subjects while under the transparent “hood”.   
Surveys: There are no risks involved beyond what would reasonably be encountered in 
everyday life. It is possible that participants may experience the following during 
completion of surveys: 
1. 
Loss of privacy as a result of a breach of confidentiality  
2. 
Undue stress related to content on the survey instruments 
 
Mitigation of Risks  
 
Dual Energy X-ray Absorptiometry (DEXA): A urine pregnancy test will be done prior 
to scans of all women of childbearing potential (all women except those with prior 
hysterectomy, tubal ligation, or absence of menses for >2 years). 
 
Magnetic Resonance Imaging: There will be a strict safety screening protocol, to ensure 
any people with contraindications are excluded from volunteering.  
 
Incidental Findings: There will be no diagnostic analysis associated with any of the MR 
sequences used in this protocol.  However some of the MR images we obtain as part of this 
protocol may show incidental medical findings.  In the case where a medical abnormality 
is apparent on an image, the image will first be reviewed by an investigator on this protocol.  
If the abnormality is confirmed, then the participant will be instructed to seek medical 
attention from their health care provider.       
  
Maximal Oxygen Consumption (VO2max) test and exercise training:  Prior to conducting 
any exercise testing, a history and physical including ECG will be conducted and will be 
used to determine whether a subject is clear to participate in exercise testing.   An 
MD/DO/NP/PA will be present during exercise testing for subjects who are at risk 
according to the American College of Sports Medicine (ACSM) Guidelines. ECG, blood 
pressure, and heart rate will be monitored during the test. 
 
Hair Collection: Hair collection will be performed by trained and qualified study staff. 
  
Adipose Tissue Biopsy: Adipose biopsies will be conducted by qualified staff following 
institutional policies and procedures including sterile techniques and sterile dressing to the 
site. Prior to the biopsies, participants will be asked for allergies including lidocaine and 
the use of anticoagulants. Subjects that have an allergy to lidocaine or have used 
anticoagulants in clinically significant amounts will be excluded. Sutures will be used to 
close the site, for those with a known allergy to steri strips.  
  
Muscle Tissue Biopsy: Muscle biopsies will be conducted by qualified staff following 
institutional policies and procedures including sterile techniques and sterile dressing to the 
site. Prior to the biopsies, participants will be asked for allergies including lidocaine and 

 
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the use of anticoagulants. Subjects allergic to lidocaine or that used anticoagulants in 
clinically significant amounts will be excluded. Sutures will be used to close the site, for 
those with a known allergy to steri strips.  
  
Intravenous lines/blood draws (lab samples, e.g.): All venipuncture will be conducted 
by qualified staff using aseptic techniques.    
  
Subcutaneous microdialysis: Insertion will be conducted by qualified staff using aseptic 
technique to reduce these risks.  
  
Deuterium-labeled water (2H2O) administration: Separated doses (35 mL) for 2H2O 
consumption will minimize symptoms.  
  
Hyperinsulinemic-euglycemic clamp:   
It is very unlikely that severe hypoglycemia will occur during the insulin infusion because 
blood glucose will be monitored every 5 to 10 minutes at the bedside and glucose infusions 
of 20% dextrose will be adjusted to maintain euglycemia (plasma glucose ~ 90 mg/dl). The 
participant is also kept awake at all times during the insulin infusion so that they can report 
or be observed for any changes. If the participant’s blood glucose is nearing hypoglycemic 
levels, the plasma glucose will be checked even more frequently, and the 20% dextrose 
infusion rate increased. If hypoglycemia occurs, the insulin infusion will be discontinued, 
and intravenous glucose continued at higher rates along with the intake of oral 
carbohydrates.  The volunteers will then be monitored for at least 2 hours during the IV 
glucose infusion and oral supplementation, then for at least 1 hour after discontinuing the 
IV glucose.  Additionally, there will be in-house supervision by a qualified physician or 
mid-level provider at all times during the procedure and immediate availability of 
corrective measures (IV 50% dextrose and/or glucagon). 
  
 
Resting Metabolic Rate/Respiratory Quotient (RMR/RQ): A member of the study 
staff will remain with the subject at all times to ensure his/her comfort. 
 
Surveys: Surveys will be offered via password protected computer-based software 
REDCap. IP addresses will not be collected. Survey results will be aggregated, no 
individual responses will be shared. Participants may opt out of survey questions they 
develop discomfort with. 
 
Provisions to Protect the Privacy Interest of Subjects 
 
Participants will be assigned unique identifiers for study-related records. All precautions 
will be taken to make sure that only authorized individuals will access participant 
research records.  The collection of sensitive information about participants will be 
limited to the minimum necessary to achieve the aims of the research, so that no 
unneeded sensitive information will be collected. 
 

 
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Early Withdrawal of Subjects 
Investigator Withdrawal of Subjects 
 
The participation in this study may be stopped at any time by the study PI without the 
participant’s consent because:  
• 
The study medical investigator thinks it necessary for subject’s health or 
safety;  
• 
Participant has not followed study instructions;  
• 
The AdventHealth Translational Research Institute has stopped the study;  
• 
Administrative reasons require the participant’s withdrawal;  
• 
Participant is unable to complete a procedure;  
• 
We are unable to place a venous line or place a catheter for the subcutaneous 
microdialysis.  
 
Subject Request for Withdrawal from Study 
 
Participation in this study is voluntary. Participants may decide not to participate in this 
study or may withdraw from this study at any time without penalty or loss of benefits. If a 
participant leaves the study before the final regularly scheduled visit, she/he may be 
asked by the study doctor to make a final visit for some ‘end-of–study’ procedures. This 
is to make sure that there are no safety concerns 
 
Data Collection and Follow-up for Withdrawn Subjects 
 
Participants who request withdrawal or who are withdrawn by the PI from the study will 
have their data maintained in the research database up to the point of withdrawal. The 
available data will be included in subsequent analysis because keeping these participants 
in the analysis is essential for study validity. 
 
Adverse Events - Recording and Reporting 
 
Each participant will be evaluated for adverse events at every study visit. Any event that is 
reported to the study staff and which meets the criteria of an adverse event will be 
documented as such and graded as to its attribution (unrelated to protocol, or possibly, 
probably, or definitely related to protocol) and severity (mild, moderate, or severe). Any 
severe and/or unanticipated adverse event will be immediately reported to the IRB 
according to AdventHealth IRB guidelines.  
  
At each contact with the subject, the study team will seek information on adverse events 
by specific questioning and, as appropriate, by the Provider through 
examination.  Information on all adverse events will be recorded immediately in the 
source document, also in the appropriate adverse event module of the case report form 
CRF.  All clearly related signs, symptoms, and abnormal diagnostic procedures results 

 
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will be recorded in the source document. All adverse and unexpected events will be 
reported according to AdventHealth IRB guidelines.  
 
Safety Monitoring Plan   
 
Safety Monitoring 
 
Adverse events will be documented and reported by the study coordinator, Medical 
Investigator or other TRI/PBRC staff. Research and safety data will be reviewed by the 
PI. This review will take place at regular meetings with the research coordinator and 
Medical Investigator. Other items discussed will include progress or adverse events 
occurring in the following: participant confidentiality, participant recruitment, and 
consent process. Both the TRI and PBRC have a standing committee at each institution 
that meets monthly to review all adverse events in the clinical trials and will additionally 
be charged with review of the study. 
 
A Data and Safety Monitoring Plan (DSMP) Committee will also be established. A group 
of researchers at AH TRI and PBRC will oversee the DSMP. It will include: 
1. MPIs: Lauren Sparks, Ph.D. and Ursula White, Ph.D. 
2. MIs: Frank Greenway, M.D. and non-MD Provider (PA or ARNP level) 
3. Biostatisticians: Robbie Beyl, Ph.D. and Fanchao Yi, M.S. 
4. Study Coordinators (TBD) 
5. Independent Safety Monitor (TBD) not directly involved in the study 
The DSMP Committee meetings may be conducted in-person, via conference call, or 
webinar. The main focus will be on participant accrual, protocol compliance, review of 
lab safety data, and review of all AEs/SAEs. No outcome data/results will be discussed 
until after completion of the data collection. The DSMP committee will meet once at 
least annually and an ad hoc meeting will be held with the Mis in case of a serious 
adverse event or death. Minutes will be recorded and forwarded to all members of the 
committee. The Independent Monitor will review and identify any deficiencies in the 
DSMP and provide the results of the reviews and advise guidance and recommendations 
concerning the study. Following each meeting, the DSMP committee will provide written 
documentation regarding findings for the study as a whole and any relevant 
recommendations related to continuing, changing, or terminating the study. A copy will 
be provided to the IRB. 
 
Data and Safety Monitoring Board (DSMB) or Equivalent 
 
 
N/A 
 
Ethical Considerations 
 

 
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Participation in this study is voluntary. Subjects may decide not to participate in this study or 
may withdraw from this study at any time without penalty or loss of benefits. No vulnerable 
populations will be studied in this protocol.  
 
Sharing of Results with Subjects: Participants will be offered the opportunity to meet with 
the Principal Investigator or designated medical staff to review the results of their lab 
assessments or other standard clinical data. Copies of their testing results will be made 
available to the participants upon request. In addition, the Principal Investigator or 
designated study staff will provide an overview of the study's outcome to the participant if 
he or she requests the information.  
 
Funding Source 
 
This study will be supported by a research program grant (RPG) from the National Institutes of 
Health, National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK133529, P.I. 
Lauren M. Sparks, PhD). The IRBNet # for this grant is 1949440.  
 
Subject Stipends or Payments 
 
Participants at TRI may receive up to $1,000 for completion of all procedures. If participant is 
unable to complete all study visits, the stipends will be prorated to the following:    
   
Visit   
Prorated amount   
Screening visit  
$25  
Visit 1 & 2  
$200  
Muscle Biopsy (Optional) 
$50 
12-week intervention   
$450  
Visit 3 & 4  
$225  
Muscle Biopsy (Optional) 
$50 
Total for all completed visits   
$1,000  
   
Participants at PBRC may receive up to $1,000 for completion of all procedures. If participant is 
unable to complete all study visits, the stipends will be prorated to the following:   
  
Visit   
Prorated amount   
Visit 1 & 2  
$200  
12-week intervention  (Weeks 1, 3, 5, 7, 9, 
11) 
$50 biweekly 
Visit 3 & 4  
$500 
Total for all completed visits   
$1,000 
 

 
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Payment for each visit is contingent on completing the visits and procedures listed above. A 
prepaid card will be the method of payment and will be requested upon completion of each study 
visit/phase or on their last day of participation, should they find they cannot complete the study.     
   
Participants’ payment will be requested within 3 business days from completion of each study 
visit/phase.   
   
For participants who are not AdventHealth Employees   
 
If participants receive more than $600 in payments in a calendar year from AdventHealth, this 
income will be reported to their IRS. The participant may be required to pay tax on this income.   
   
For participants who are AdventHealth  or AdventHealth Medical Group Employees   
   
All payments will be reported as added income to the participant’s base salary and will be taxed 
on a future paycheck. If the participant is an employee of AdventHealth, their decision to 
participate will not affect their employment or relationship with AdventHealth.   
 
Publication Plan 
 
We attest that the TRI faculty and staff will adhere to POLICY-TRI-ADM-005 (Access to 
Clinical Trial Data for Publication Purposes). Assignment of authorship and the contributions of 
each author will be determined by the International Committee of Medical Journal Editors 
(ICMJE) policy to guide authorship. 
   
 
 
 
References 
 
1.  
Ryden, M., D.P. Andersson, and P. Arner, Usefulness of surrogate markers to determine 
insulin action in fat cells. Int J Obes (Lond), 2020. 44(12): p. 2436-2443.  
2. 
Sparks, L.M., et al., Relation of adipose tissue to metabolic flexibility. Diabetes Res Clin 
Pract, 2009. 83(1): p. 32-43.  
3. 
Allerton, T.D., et al., Exercise reduced the formation of new adipocytes in the adipose 
tissue of mice in vivo. PloS one, 2021. 16(1): p. e0244804.  
4. 
White, U.A., et al., Differences in In Vivo Cellular Kinetics in Abdominal and Femoral 
Subcutaneous Adipose Tissue in Women. Diabetes, 2016. 65(6): p. 1642-7.  
5. 
White, U.A., et al., Association of In Vivo Adipose Tissue Cellular Kinetics With Markers 
of Metabolic Health in Humans. J Clin Endocrinol Metab, 2017. 102(7): p. 2171-2178.  
6. 
Corvera, S., Cellular heterogeneity in adipose tissues. Annual review of physiology, 
2021. 83: p. 257-278.  
7. 
Lee, Y.H., A.P. Petkova, and J.G. Granneman, Identification of an adipogenic niche for 
adipose tissue remodeling and restoration. Cell Metab, 2013. 18(3): p. 355-67.  

 
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8. 
Whytock, K.L., et al., Single cell full-length transcriptome of human subcutaneous 
adipose tissue reveals unique and heterogeneous cell populations. iScience, 2022. 25(8): 
p. 104772.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Appendix: 
 
The following table identifies the procedures in relation to the study timeline for CTRL group: 
 
 
Screen 
Baseline 
2H2O Intervention Period 
Follow-up 
 
 
 
Week 
 
 
Visits 
SV1 
V1 
V2 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
V3 
V4§ 
Days 
 
-14 
-8 
-7 
0-6 
7-13 
14-20 
21-27 
28-34 
35-41 
42-48 
49-55 
56-62 
63-69 
70-76 
77-83 
85 
87 
88 
Windows 
Within 28 
days of 
V1 
±2 
±2 
 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility Assessment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Collection (urea, nitrogen 
and creatinine) 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Urine Collection (pregnancy, if 
applicable) 
X 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
 
Urine Collection (toxicology) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam + Medical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital Signs 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anthropometrics 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Screening Blood Draw 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Electrocardiogram 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Surveys 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
MRI 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
DEXA 
 
X 
 
 
 
 
 
 
X* 
 
 
 
 
 
 
 
X 
 
 
VO2max Test 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Overnight Stay with 
Standardized Meal 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
7-Day Food Record Start 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
7-Day Food Record Return 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Hair Collection 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
WAT Biopsy 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Muscle Biopsy 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Respiratory Quotient (Hood) 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Pre-Clamp Blood Draw 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 

 
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Hyperinsulinemic-euglycemic 
Clamp + Microdialysis 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Accelerometer Provided 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Biopsy and Catheter Insertion 
Site Check 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
On-site Body Weight 
Measurement 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
Weight Maintenance and Health 
Education 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
2H2O Pick-up 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
2H2O Container Return 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
 
2H2O Urine Collection 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Review Adverse Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
*DEXA can take place between Weeks 4-6 to increase scheduling flexibility 
§Visit 4 procedures must be done within 36-48 hours after the VO2max test 
On-site Body Weight Measurement, Weight Maintenance and Health Education, and 2H2O activities must occur on the first day of exercise 
for that week. 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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The following table identifies the procedures in relation to the study timeline for EX group: 
 
Screen 
Baseline 
2H2O and Exercise Intervention Period 
Follow-up 
 
 
 
Week 
 
Visits 
SV1 
V1 
V2 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
V3§ 
V4 
Days 
 
-14 
-8 
-7 
0-6 
7-13 
14-20 
21-27 
28-34 
35-41 
42-48 
49-55 
56-62 
63-69 
70-76 
77-83 
85 
87 
Windows 
Within 28 
days of 
V1 
±2 
±2 
 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
±1 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility Assessment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Collection (urea, nitrogen 
and creatinine) 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Urine Collection (pregnancy, if 
applicable) 
X 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
 
Urine Collection (toxicology) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam + Medical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital Signs 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anthropometrics 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Screening Blood Draw 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Electrocardiogram 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Surveys 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
MRI 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
DEXA 
 
X 
 
 
 
 
 
 
X* 
 
 
 
 
 
 
 
X 
 
 
VO2max Test 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Overnight Stay with 
Standardized Meal 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
7-Day Food Record Start 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
7-Day Food Record Return 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Hair Collection 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
WAT Biopsy 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Muscle Biopsy 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Respiratory Quotient (Hood) 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Pre-Clamp Blood Draw 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Hyperinsulinemic-euglycemic 
Clamp + Microdialysis 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Accelerometer Provided 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Biopsy and Catheter Insertion 
Site Check 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 

 
Current Version Date: 
2/5/2024 
IRBNet #: 
1805407 
  
 
Page 42 of 42 
Research Study protocol template version date 10/05/2022 
 
Supervised Exercise Training 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
On-site Body Weight 
Measurement 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Weight Maintenance and Health 
Education 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
2H2O Pick-up 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
2H2O Container Return 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
 
2H2O Urine Collection 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Review Adverse Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
*DEXA can take place between Weeks 4-6 to increase scheduling flexibility 
§Visit 3 must be done 48-72 hours after the last exercise session. Visit 4 must be done within 36-48 hours after the VO2max test. 
On-site Body Weight Measurement, Weight Maintenance and Health Education, and 2H2O activities must occur on the first day of exercise 
for that week. 
Weight Maintenance and Health Education will occur as needed following body weight measurement.