Abscisic Acid Effects on Glucose Homeostasis and Insulin Sensitivity
Phase: PHASE2
Conditions: Pre Diabetes
Sponsor: AdventHealth Translational Research Institute

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 1 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Study Protocol Title: 
ABA - Abscisic Acid Effects on Glucose Homeostasis and Insulin Sensitivity 
 
Study Sponsor:   
Biotherapeutics Inc (BTI), a spinoff Company of Virginia Tech 
 
Principal Investigator: 
Principal investigator: Bret Goodpaster, PhD 
 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 2 of 38 
Research Study protocol template version date 08/07/2020 
 
 
List of Abbreviations: 
AE 
Adverse Event 
ANCOVA 
Analysis of Covariance 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
DRE 
Disease-Related Event 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act of 2007 
FFR 
Federal Financial Report 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intention-To-Treat 
LSMEANS Least-squares Means 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NIH IC 
NIH Institute or Center 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
QA 
Quality Assurance 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 3 of 38 
Research Study protocol template version date 08/07/2020 
 
 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
T2D 
Type 2 Diabetes/Diabetics 
UP 
Unanticipated Problem 
US 
United States 
 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 4 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Introduction 
 
This document is a protocol for a human research study. This study is to be conducted in 
accordance with applicable federal regulations and institutional research requirements and 
procedures. 
 
The purpose of this study is to determine the effects of oral abscisic acid (ABA) on glucose 
metabolism in subjects with defined prediabetes and type 2 diabetes (T2D). To this end, a 
randomized, cross-over, placebo-controlled trial will be employed to evaluate the effects of ABA 
(95ug) twice a day (190ug total per day) for 14 days on tolerability and insulin sensitivity during 
a hyperinsulinemic euglycemic clamp.  Participants will be randomized to either ABA (95 µg 
ABA and 300 mg of corn starch) or placebo (300 mg cornstarch) twice a day before meals for 14 
days. 
 
The primary aim is to determine the tolerability and efficacy of ABA supplementation on insulin 
sensitivity in prediabetic and T2D men and women. The secondary aim is to assess changes in 
insulin-stimulated signaling and glucose transporter proteins, insulin, glucose, pyruvate, and fatty 
acid oxidation levels in skeletal muscle tissue samples from biopsy in addition to inflammatory 
markers and fasting ABA concentrations in plasma.  
 
Background Information and Scientific Rationale 
 
Prediabetes afflicts 86 million Americans and has recently risen to more than 3 million new 
cases per year in the U.S. alone. A prediabetic person is likely to become diabetic within 10 
years, because many people with prediabetes have no symptoms and do not adhere to lifestyle 
and diet changes or medical treatment. According to the CDC, 9 out of 10 Americans with 
prediabetes are not aware that they have it. Furthermore, approximately 1 in 10 Americans (~37 
million) have type 2 diabetes. Insulin resistance within skeletal muscle is considered to be a key 
instigator of prediabetes and T2D. Fortunately, skeletal muscle insulin resistance is reversible 
and/or mitigated with lifestyle changes and the right diet; it is possible to restore andor manage 
glucose homeostasis. There are currently no specific drugs that target skeletal muscle insulin 
sensitivity. Identifying natural ingredients that can be produced and marketed to enhance insulin 
sensitivity could significantly impact the nearly 110 million people in the U.S. with either 
prediabetes or T2D. 
 
Abscisic acid (ABA) has been identified as a putative sugar control hormone. The plasma 
ABA concentration (ABAp) increases after OGTT in healthy subjects (1). ABA stimulates 
glucose-dependent insulin release from human and rodent pancreatic β-cells (2). It increases both 
translocation of the glucose transporter GLUT-4 to the plasma membrane and GLUT-4-
dependent glucose uptake by cultured adipocytes and myoblasts (3). The increase of ABAp that 
occurs after an OGTT is impaired in patients with T2D and in women with gestational diabetes 
(GDM) (4). Glucose tolerance after childbirth is paralleled by restoration of both the ABAp 
response to oral glucose and normal fasting ABAp levels (4). The impaired response of ABAp to 
hyperglycemia is a common feature in T2D and prediabetic subjects, pointing to a critical role 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 5 of 38 
Research Study protocol template version date 08/07/2020 
 
 
for ABAp in maintaining glycemic control. Based on studies in animals and humans (5), 
developing an ABA-based medical food to help prediabetic individuals may be highly beneficial. 
 
Significance of generating GRAS (Generally Recognized as Safe) ABA. GRAS is a strict 
safety standard established by the FDA, requiring technical demonstration of non-toxicity and 
safety, as well as a general recognition and agreement by experts that the ingredients are safe for 
consumption. The FDA has determined many ingredients are GRAS; they are listed as such by 
regulation, in Volume 21 Code of Federal Regulations (CFR) Sections 182, 184, and 186. Other 
ingredients may achieve self-affirmed GRAS status via a panel of independent experts in the 
pertinent field who co-author a GRAS report. The process of attaining GRAS status is one of 
self-determination, whereby a panel of experts (the Expert Panel) critically evaluates the 
documentation supporting the safety of the ingredient under the conditions of intended use 
before providing a signed opinion. The material reviewed by the Expert Panel includes 
information pertinent to product manufacturing, specifications, and analyses of the substance, 
proposed uses and levels of use in foods, estimated dietary exposure, safety studies, and other 
information to support safety, as available. Following a successful GRAS determination, the 
ingredient can be marketed immediately.  
 
Many U.S. food manufacturers will only purchase foods/beverages if they have, at a minimum, 
an FDA “No Objection” letter to a GRAS Notification. Regardless of the pathway to GRAS, the 
manufacturer maintains documentary evidence of the safety of the ingredient, including its 
natural or artificial derivation, the related processing, and the effects of its consumption under 
specified intended uses. Examples of successful commercial development of natural products as 
medical foods include: 1) Ensure® and Glucerna® for glucose control (Abbott), 2) Axona® for 
Alzheimer’s patients, 3) Fosteum® (genistein aglycone/citrated zinc bisglycinate/cholecalciferol) 
for osteopenia and osteoporosis, 4) Metanx® (l-methylfolate calcium/pyridoxal 5′-
phosphate/methylcobalamin) for diabetic neuropathy, and 5) TheramineTM (l-arginine, 5-htp, 
histidine, l-glutamine) for myalgia. During the Phase 1 SBIR period, Biotherapeutics Inc (BTI) 
has successfully secured GRAS for ABA in October 2016 and is planning to use this product for 
clinical testing and commercialization. This Phase 2 proposal will generate qualified health 
claims through translational and clinical studies that allow developing ABA as a functional food 
ingredient for managing blood sugar levels. 
 
ABA is an innovative medical food for metabolic syndrome, prediabetes, and T2D. The first 
line of management for prediabetes, metabolic syndrome, and diabetes includes diet and 
exercise; however, patient adherence is exceedingly difficult. Therapeutic alternatives are 
increasingly preferred, especially options that do not require a clinical visit for administering 
treatment. Thus, a medical food for glycemic control and treating T2D is desirable. Further, 
developing a medical food that exerts anti-diabetic and anti-inflammatory effects with the 
potential to prevent prediabetes and metabolic syndrome is novel.  Moreover, ABA, a fruit-
derived compound, is well suited to be developed into a medical food. ABA obtained from 
normal dietary consumption (3)) is insufficient to exert glycemic control. U.S. adults with 
metabolic syndrome and prediabetes have suboptimal ABAp levels, thus there might be an 
unmet nutritional need for increasing ABA intake. Marketing ABA as a medical food 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 6 of 38 
Research Study protocol template version date 08/07/2020 
 
 
formulation, as opposed to a pharmaceutical, could increase patient interest and the overall 
public health impact of this product. Preclinical and clinical studies have revealed an outstanding 
safety profile for ABA at 10,000, 15,000 and 20,000 ppm. ABA stimulates glucose consumption 
by adipocytes and L6 myoblasts, and lowers glycemia in humans when supplemented with 1 
µg/kg body weight (6). We validated that low-doses of ABA (1, 10, 40 and 200 µg/kg/d) 
significantly reduce fasting blood glucose and A1C levels in severely diabetic db/db mice when 
administered daily for 28 d. In two independent preliminary human Phase 1 studies, a single dose 
of ABA administered concurrently with a caloric load improved glucose tolerance and reduced 
glycemic and insulin indices in healthy subjects. This project will move ABA to the forefront of 
translational and clinical research on metabolic syndrome, prediabetes, and diabetes. 
 
ABA is an innovative ingredient that offers a natural solution to high sugar levels in blood. 
ABA is present in varying concentrations in fruits and vegetables. On average the concentration 
of ABA is 1.54 per mg/kg of wet weight of vegetable and 0.49 mg/kg of wet weight of fruit. 
According to the latest published dietary survey data from 2009–2012 (7), U.S. adults age ≥20 
years consumed on average 2.79 servings/day of fruits and vegetables. The current 
recommendation is to eat ≥4.5 servings per day, which would lead to ≥718 µg/d of ABA in an 
ideal situation, but only 8% of the U.S. adult population is meeting the AHA 2020 Strategic 
Goals on dietary recommendations for fruits and vegetables (7). This means that 92% of the 
population might be obtaining less ABA from the diet than they should according to the fruit and 
vegetable recommendations. The addition of 2 doses of 95 μg each  (total 190 μg/day) should 
bring plasma ABA levels closer to the recommendations for fruit and vegetable intake. Further, 
given its impact on glycemic control, a need to develop reference values for ABA exists. 
 
LANCL2 is the proposed molecular target for the glycemic-controlling effects of ABA. The 
LANCL2 is a unique pathway that is not being exploited commercially by any other anti-diabetic 
medications. The mammalian ABA receptor has been proposed to be LANCL2 on the basis of, i) 
molecular modeling predictions (8), ii) direct and specific ABA binding to the purified 
recombinant protein (9), and iii) abrogation of the functional effects of ABA by silencing of 
LANCL2 expression in ABA-sensitive cells and deletion of the gene in mice. The signaling 
pathway downstream of LANCL2 includes a G-protein-mediated activation of adenylate cyclase, 
cAMP production and activation of protein kinase A (10). In addition, LANCL2 facilitates 
phosphorylation of Akt by mTORC2 via direct physical interactions (11). Active mTORC2 
causes translocation of GLUT4 to the plasma membrane and stimulates glucose uptake (12). 
Increased Akt phosphorylation, GLUT4 translocation and GLUT4-dependent glucose transport 
occur in ABA-treated L6 myoblasts (3). Thus, the ABA/LANCL2 axis is emerging as a target for 
treating dysfunctional glucose homeostasis (13). ABA performs within 98% similarity to the 
type 2 diabetes drug, Avandia®, a PPAR gamma-agonist.  Additionally, as opposed to TZDs, 
ABA does not bind to PPAR gamma (14). Biochemical analyses validated our finding that 
LANCL2 recognizes ABA (9, 10, 14).  
 
LANCL2 expression in immune cells, skeletal muscle, adipose, and pancreas, and the potential 
to manipulate LANCL2 signaling and GLUT4 translocation with ABA make it a putative 
therapeutic and/or adjunct for metabolic syndrome, prediabetes and T2D. BTI has contributed to 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 7 of 38 
Research Study protocol template version date 08/07/2020 
 
 
elucidating the role of LANCL2 in inflammation and glucose metabolism. BTI performed the 
initial modeling studies and predicted that ABA binds to LANCL2 (8). This prediction was 
validated by biochemical assays (8, 9). We discovered that dietary ABA increases insulin 
sensitivity and suppresses obesity-related inflammation in obese/diabetic db/db mice (15, 16). 
Furthermore, µg amounts of ABA from fruit extracts improve glucose tolerance while also 
reducing insulinemia in both rats and humans (6, 17). Moreover, we provide mechanistic 
evidence in vivo that the molecular target of ABA might be LANCL2, as the loss of LANCL2 
expression in LANCL2-/- mice diminishes the glucose-normalizing effects of oral ABA 
treatment. ABA increased glucagon-like peptide-1 (GLP1) levels in wild-type (WT) mice, 
stimulated GLP1 secretion from L-cells, and increased plasma GLP1 levels in rats (1). However, 
the loss of LANCL2 results in increased plasma insulin levels and decreased GLP1 levels in 
acute high fat feeding study.  
 
BTI Secures GRAS for ABA. An independent expert panel using scientific procedures (U.S. 
FDA, 1997) determined that ABA, intended for use in certain foods within the general categories 
of functional beverages, foods and fruit juices, at a level of up to 95 µg/6-12 fl oz twice a day, is 
(i) exempt from the premarket approval requirements of the Federal Food, Drug, and Cosmetic 
Act and (ii) is GRAS for such use. Given that ABA is naturally occurring in food, oral exposure 
to ABA at food comparable levels is not expected to cause toxicological effects. Consistent with 
its nature as a food component, ABA was well-tolerated and there were no signs of toxicity in 
several dietary studies with ABA in mice, rats, and humans. ABA was negative in an in vitro 
reverse mutation assay in bacteria, an in vitro chromosome aberration assay in Chinese hamster 
ovarian cells and an in vivo mouse micronucleus assay. ABA is not a developmental toxicant in 
rats. A two-generation reproduction study shows that ABA: 1) does not cause detriment to the 
reproduction rate or efficacy in rats and 2) does not cause numbers or follicle counts to regress. 
In sub-chronic toxicity studies following a 4-week and a 13-week dietary intervention with 
different concentrations of ABA in rats, no adverse toxicological effects were seen for 90 d at 
intakes of up to 20,000 ppm (around 1,500 mg/kg bw/d). Therefore, ABA has a no observable 
adverse effect level (NOAEL) of 20,000 ppm which corresponds to 1,500 mg/kg body weight in 
mice. ABA has an acute oral median lethal dose (LD50) of more than 5,000 mg/kg body weight 
in mice. Furthermore, ABA is recognized as a substance in which a maximum permissible level 
is not applicable as no signs of toxicity have been observed in a multitude of toxicity studies. 
Pharmacokinetic (PK) analyses of ABAp levels following oral and i.v. administration 
demonstrate that ABA is systemically distributed has high bioavailability (BA). Specifically, a 
BA study comparing the area under the curve of plasma concentrations after oral and I.V. 
administration of 10 µg/kg gave a BA of >65%. Based on this PK profile of ABA, we 
determined the effects of one versus two doses of ABA daily. The latter provided superior 
glucose normalizing effects in db/db mice. Our ABA source is extracted from figs has shown 
efficacy and safety in mice and humans. 
 
The scientific impact of this project is to develop the first ABA-containing medical food for 
glycemic control. We will conduct the translational and clinical studies that will provide the 
scientific basis for qualified health benefits of ABA in glycemic control. 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 8 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
Study Objectives 
 
Primary Objective/Aim/Goal/Hypothesis 
 
The primary objectives are to assess the tolerability (number of adverse events) and 
efficacy of ABA on insulin sensitivity compared to placebo. 
 
Secondary Objective/Aim/Goal/Hypothesis 
 
Secondary objectives are to assess changes in insulin stimulated signaling and glucose 
transporter proteins in muscle biopsies, along with insulin levels, glucose, pyruvate, and 
fatty acid oxidation in skeletal muscle tissue samples, inflammatory markers and fasting 
ABA concentrations in plasma. 
 
Study Design 
 
Research Design 
 
We propose to conduct a randomly assigned cross-over double-blind placebo-controlled 
trial (RCT) to evaluate the tolerability of ABA supplementation and efficacy of ABA to 
increase insulin sensitivity in subjects with prediabetes and T2D.  Subjects will be 
randomly assigned to one of two groups: (1) ABA (95 ug 2x/day), or (2) Placebo (Corn 
Starch 300 mg) for 14 days. Subsequently, subjects will take part in a 4-5 week washout 
period then cross over and complete another 14-day treatment in the opposite study arm. 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 9 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
 
 
 
 
 
 
Screening  
Visit 
 
 
Visit 1 
 
 
Visit 2 
 
 
 
 
 
 
 
 
 
Visits 3-7 
 
 
 
Visit 8-9 
 
 
 
  
 
 
 
 
Visit 10 
 
 
Visits 11 
 
 
 
Total N=24:  Obtain informed consent. Screen potential participants by inclusion and exclusion criteria; 
obtain health history and physical, and basic anthropometrics; blood and urine collection. For additional 
details of the trial see Section 1.3, Schedule of Activities 
Placebo 
N=12 
ABA 
N=12 
Randomize 
Participants will complete a DEXA and questionnaires to assess physical activity and diet. 
Participants will arrive to TRI-MD for an overnight stay. The following morning participants will complete a 
4hr hyperinsulemic euglycemic clamp, muscle biopsies, indirect calorimetry, and blood draws. 
Participants will undergo 14 days of supplementation 
ABA: 95µg 2x/day 
Placebo: Corn Starch 300mg 
Participants will arrive to TRI-MD for an overnight stay. The following morning participants will 
complete a 4hr hyperinsulemic euglycemic clamp, muscle biopsies, indirect calorimetry, and blood 
Placebo 
N=12 
ABA 
N=12 
Cross-Over Supplement Following a Wash-Out 
Participants will arrive to TRI-MD for an overnight stay. The following morning participants will 
complete a 4hr hyperinsulemic euglycemic clamp, muscle biopsies, indirect calorimetry, and blood 
Participants will complete a DEXA, and questionnaires to assess physical activity and diet. 
Participants will undergo 14 days of supplementation 
ABA: 95µg 2x/day 
Placebo: Corn Starch 300mg 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 10 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Visits 12-16 
 
 
 
Visit 17 
 
Visit 18 
 
Research Intervention Description  
 
Pharmacologic Intervention 
 
This is a Phase 2 single-site study. The study population will include males and females of all 
races and ethnicities,18-75 years of age from the greater Orlando metropolitan areas who are pre 
diabetic (fasting glucose 100-125mg/dl/ 5.5-6.9mmol) and/or diagnosed T2D.   
This is a randomly assigned cross-over double-blind placebo-controlled trial (RCT) design.  As 
such, subjects will be randomly assigned to one of the groups (ABA or placebo) for 14 days, 
then after a 4-5 week washout period, will complete another 14-day treatment in the opposite 
study arm. 
Subjects in the ABA study arm will be supplemented with ABA at a dose of 95 ug twice per day. 
ABA is a GRAS (generally recognized as safe) ingredient being developed for inclusion within 
health foods designed for improved glucose homeostasis. The placebo group will consume a 
control supplement (corn starch) 300 mg twice per day. 
 
Drug preparation 
 
Both study products (ABA and placebo) used for this trial will be packaged and clinically 
labeled by Biotherapeutics, Inc.  The control and experimental products will be identified using a 
unique study number for randomization purposes, however all parties involved in the study will 
remain blinded.  Both products will be delivered to the Research Pharmacy at the TRI. 
 
Purified ABA is a white, crystalline solid. ABA may be isolated and enriched from fruits and 
vegetables, particularly apricots and figs. ABA is a small isoprenoid compound of 264.32 g/mol 
molecular weight. It has a boiling point of 160-161°C. ABA presents no health, fire or reactivity 
hazards and is recognized as safe for consumption by the FDA. ABA and placebo will be 
administered as a powder in capsules.  
 
Study products are to be stored in an access-controlled environment at ambient temperature 
under conditions suitable for food storage (i.e., sanitary conditions, off the floor and organized to 
minimize the risk of product dispensing errors).  The investigator agrees not to supply study 
product to any person not enrolled in the study or to any physician or scientist not named as an 
investigator.  The investigator also agrees to use the study product only within the context of the 
Participants will arrive to TRI-MD for an overnight stay. The following morning participants will 
complete a 4hr hyperinsulemic euglycemic clamp, muscle biopsies, indirect calorimetry, and blood 
Participants will return to TRI-MD to assess the sites where the muscle biopsies were taken. 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 11 of 38 
Research Study protocol template version date 08/07/2020 
 
 
study protocol.  Subjects will be asked to return any study product remaining after the 
completion of the study to the investigator.   
 
Biotherapeutics, Inc will review the investigator’s product accountability records.  The 
investigator will return to Biotherapeutics, Inc or destroy [render product unusable by destroying 
primary packaging (i.e. packaging in direct contact with product)] all unused product after all 
study supply inventories have been reconciled at the conclusion of the study. 
 
This study is a double blinded study.  Neither the investigator(s), their staff, appropriate member 
of Biotherapeutics, Inc. staff, or subjects will be informed of the identity of any of the study 
products during the clinical portion of the study.  The study center personnel will not analyze the 
contents of the study products or in any way seek to learn the identity of the study products.  The 
investigator should ensure that if it becomes necessary, blinding is broken only in accordance 
with the protocol. 
Each subject should consume two doses of product per day.  Product will be administered and/or 
distributed by study staff and recorded on product consumption forms by study staff and 
subjects.   
Study Site(s)/Location(s) and Number of Subjects 
 
AdventHealth Orlando site locations (campus, physician offices, etc): Translational 
Research Institute 301 E. Princeton St., Orlando, Florida 32804 
Estimated number of subjects at AdventHealth Orlando sites: 24 
 
Total number of all sites: 1 
Estimated number of subjects at all sites combined: 24 
 
 
Multi-Site Research Logistics/Communication Plan 
 
N/A 
 
Research Conducted in a Foreign Country 
 
N/A 
 
Community-Based Participatory Research 
 
N/A 
 
Subject Selection 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 12 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Vulnerable Populations (if applicable) 
 
AdventHealth Employees: Recruitment efforts will follow AdventHealth recruitment Standard 
Operating Procedures (SOPs) for research.  AdventHealth employees will not be individually 
targeted nor excluded from study participation based on employment.  AdventHealth employees 
who engage the AdventHealth Translational Research Institute 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. 
 
Inclusion Criteria 
 
1. Provision of signed and dated informed consent form 
2. Stated willingness to comply with all study procedures and availability for the 
duration of the study  
3. Male or female, aged 18-75 
4. In good general health as evidenced by medical history 
5. Fasting glucose >/= 5.6 mmol/L (100 mg/dL) and/or HbA1c >/= 5.7 
6. Ability to take oral medication and be willing to adhere to the study intervention 
regimen 
7. Females currently on hormone replacement therapy (HRT) can participate in the 
study if they have been on HRT for at least 6 months and will continue to be on HRT 
during the study. 
8. For males of reproductive potential: use of condoms or other methods to ensure 
effective contraception with partner 
9. Agreement to adhere to Lifestyle Considerations throughout study duration. 
 
Exclusion Criteria 
 
1. 19<BMI>45.0 kg/m2 
2. Currently taking insulin 
3. Blood pressure (BP) ≥ 150mmHg systolic and ≥95 mmHg diastolic 
4. Current use of medications or antioxidant vitamins or supplements that would 
impact dependent variables, including glucose metabolism. 
5. Past or current ischemic heart disease, stroke, respiratory disease, neurological 
disease, hematological-oncological disease and free of recent infection (prior 2 weeks) 
6. Pregnancy or lactation 
7. Treatment with another investigational drug or other intervention within 1 year. 
8. Current smoker or tobacco use within the past year. 
9. Disqualifying findings on physical examination include cardiac murmurs, 
diminished pulses or the presence of bruits in the lower extremities, lower extremity 
thrombophlebitis, evidence of peripheral neuropathy, paresis or edema. 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 13 of 38 
Research Study protocol template version date 08/07/2020 
 
 
10. History of a corn allergy.  
 
 
 
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.   
 
We will implement regular, ongoing discussions between the 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.   
 
With our internal database, electronic medical records and direct to consumer advertising we 
expect to recruit the required number of suitable participants within our planned enrollment period 
of approximately 24 months. The AdventHealth Translational Research Institute facilities are state 
of the art and we have within our building all 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. 
 
Study Procedures 
 
Subject Recruitment and Screening 
 
The study population will include males and females of all races and ethnicities,18-75 
years of age from the greater Orlando metropolitan areas who are pre diabetic (fasting 
glucose 100-125mg/dl/ 5.5-6.9mmol and/or HbA1C >5.7 and <6.5%) or diagnosed T2D.   
 
Recruitment methods utilized may include, but will not be limited to, the following: 
recruitment from within the AdventHealth TRI 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 Orlando IRB for review prior to using or publishing them. Recruitment 
efforts will follow AdventHealth Orlando Translational Research Institute Recruitment 
SOPs for research.  
 
Participants can be re-screened and repeat screening laboratory measurements can be 
completed at the discretion of the PI or Sub-I.   
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 14 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Consent Process  
 
We attest that all study staff delegated the authority to obtain informed consent 
will follow “Informed Consent Process and Written Documentation of Informed 
Consent” (SOP401.116). 
  
Potential subjects will be given time to read the consent document and will be interviewed 
by a clinical coordinator. The coordinator will give a detailed overview of the study and 
then ask if the potential subject has questions and if they might be interested.  The candidate 
is then encouraged to ask questions. After all questions have been addressed and interest 
remains, the candidate then signs the consent if he/she chooses to do so. After the subject 
has signed the consent, inclusion and exclusion criteria will be reviewed.  The new subject 
is given a photocopy of the signed consent form for his/her records. The PI/Sub-I will 
review all participant Inclusion Exclusion Worksheets.  Once deemed eligible, the PI/Sub-
I will sign and date the Inclusion Exclusion Worksheet.  Participants will be free to 
withdraw at any time for any reason without consequence.  
 
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)  
  

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 15 of 38 
Research Study protocol template version date 08/07/2020 
 
 
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  
 
Non-English Speaking Subjects 
 
The participant population is primarily English speaking.  In the unlikely event that a non-
English speaking participant meets criteria for enrollment, then IRB Short Form procedures 
will be initiated with the assistance of a Qualified Medical Interpreter, or Video Remote 
Interpretation will be utilized when there is a need of sight translation. A Qualified 
Bilingual Staff (QBS), or if not available, a Qualified Medical Interpreter or Video Remote 
Interpretation will be utilized if only interpreting for another staff member is required. 
 
Randomization 
 
This trial will have a triple-masked design, so that the investigators, study staff and 
the participants are masked to the drug intervention assignment. Randomization will 
be performed at each site by the investigational pharmacy based upon a 
randomization structure provided by the study biostatistician. The randomization 
scheme will be concealed to investigators and study staff until the completion of the 
study. Randomization will be performed 1:1. 
 
Only under emergent circumstances and with all appropriate approvals (i.e. study 
physician and/or DSMB) will the blinding be broken regarding the treatment group. If 
the request to unblind is granted, the ID, the reason for unblinding, the study staff 
member who performs the unblinding and a list of the persons who are not blinded will 
be recorded. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 16 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
 
 
 
Study Visits  
 
All study visits will be conducted at the AdventHealth Translational Research Institute. 
 
 
 
Baseline Period 
Intervention Period 
Follow-Up 
Period 
Drug Washout 
Period 
Visit 
SV1 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
Day 
D-28 
D-9 
D-2 
D-1 
D0 
D2 
D5 
D8 
D11 
D13 
D14 
D15 
D15-43 
Windows 
Within 
7 days 
of 
Visit 1 
±7 
 
 
±1 
±1 
±1 
±1 
±1 
 
 
±1 
±7 
HIPPA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Assessment of Eligibility 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Vital Signsa 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Weight 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Waist Circ 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Screening Blood Drawb 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysisc 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Review AEs and Con Meds 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Med Hx, Physical 
X 
 
 
 
 
 
 
 
 
 
 
 
 
DEXA 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Activity 
Questionnaired 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Activity Monitor 
One 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Physical Activity Monitor Off 
 
 
X 
 
 
 
 
 
 
 
 
 
 
4d Diet Recall 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Overnight Stay 
 
 
X 
 
 
 
 
 
 
X 
 
 
 
Hyperinsulemic Euglycemic 
Clampf 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
RMRg 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
Muscle Biopsiesh 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
Blood Drawsi 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
Randomization 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
Post Procedure Lunch 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
Biopsy Check 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Drug Washoutj 
 
 
 
 
 
 
 
 
 
 
 
X 
Drug Distributionk 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
Drug Compliance 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
Issuance of Stipend 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 17 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
 
 
 
 
 
 
 
Crossover Baseline 
Period 
Crossover Intervention Period 
Follow-Up Period 
Visit 
V10 
V11 
V12 
V13 
V14 
V15 
V16 
V17 
V18 
Day 
D44 
D52 
D53 
D54 
D56 
D59 
D62 
D65 
D67 
D68 
D69 
Windows 
±7 
 
 
±1 
±1 
±1 
±1 
±1 
 
 
±1 
HIPPA 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
 
 
 
 
 
 
 
 
 
 
 
Assessment of Eligibility 
 
 
 
 
 
 
 
 
 
 
 
Vital Signsa 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weight 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Height 
 
 
 
 
 
 
 
 
 
 
 
Waist Circ 
 
 
 
 
 
 
 
 
 
 
 
Screening Blood Drawb 
 
 
 
 
 
 
 
 
 
X 
 
Urinalysisc 
 
 
 
 
 
 
 
 
 
X 
 
Review AEs and Con Meds 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Med Hx, Physicald 
 
 
 
 
 
 
 
 
 
 
 
DEXAe 
X 
 
 
 
 
 
 
 
 
 
 
Physical Activity Questionnairef 
X 
 
 
 
 
 
 
 
 
 
 
Physical Activity Monitor Ong 
X 
 
 
 
 
 
 
 
 
 
 
Physical Activity Monitor Off 
 
X 
 
 
 
 
 
 
 
 
 
4d Diet Recallh 
X 
 
 
 
 
 
 
 
 
 
 
Overnight Stay 
 
X 
 
 
 
 
 
 
X 
 
 
Hyperinsulemic Euglycemic Clampi 
 
 
X 
 
 
 
 
 
 
X 
 
RMRj 
 
 
X 
 
 
 
 
 
 
X 
 
Muscle Biopsiesk 
 
 
X 
 
 
 
 
 
 
X 
 
Blood Drawsl 
 
 
X 
 
 
 
 
 
 
X 
 
Randomization 
 
 
X 
 
 
 
 
 
 
X 
 
Post Procedure Lunch 
 
 
X 
 
 
 
 
 
 
X 
 
Biopsy Check 
 
 
 
X 
 
 
 
 
 
 
X 
Drug Washout 
 
 
 
 
 
 
 
 
 
 
 
Drug Distribution 
 
 
 
X 
X 
X 
X 
X 
 
 
 
Drug Compliance 
 
 
 
X 
X 
X 
X 
X 
 
 
 
Issuance of Stipend 
 
 
 
X 
 
 
 
 
 
X 
 
. 
Screening Visit; Day -28 
Participants will arrive at the clinic in the morning of the screening visit (~7-8 AM) following a 
≥ 10-hour overnight fast. Participants will be encouraged to stay hydrated prior to this visit. The 
following is a list of the tests the participants will undergo after signing the Informed Consent 
Form as part of the screening procedure prior to participation in the protocol. Any participant 
who does not match the listed inclusion criteria or has any of the listed exclusion criteria (see 
above) will not be eligible to participate in the study: 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 18 of 38 
Research Study protocol template version date 08/07/2020 
 
 
aVital Signs: Blood pressure and pulse rate, respiration rate, temperature 
Height 
Weight 
Waist Circumference 
bScreening Blood Draw: Complete hemogram (RBC, Hb, MCHC, Htc, MCW, WBC, PLT), 
comprehensive chemical chemistry profile (BUN, creatinine, ALT, AST, ALP, GGT, bilirubin, 
plasma protein electrophoresis, fasting glucose, triglycerides and cholesterol, ions and anion 
gap), and HgbA1C. 
cComplete Urinalysis 
Review Adverse Events and Concomitant Medications 
dMedical History/Physical Examination: 
• Participant history: Demographics including age, marital status, education, ethnic group, 
employment status, smoking history (if applicable), and past medical history will be 
collected on a participant history form 
• Medication and Supplement Use 
Visit 1 (Day -9) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
eDEXA: 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 (all 
women except those with prior hysterectomy, tubal ligation, or absence of menses for >2 years) 
prior to the DEXA scan for safety.   
fPhysical Activity Questionnaire: International Physical Activity Questionnaire (IPAQ) 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 19 of 38 
Research Study protocol template version date 08/07/2020 
 
 
gPhysical Activity Monitor On: ActiGraph Monitor. Physical Activity Level will be measured 
objectively using an accelerometer (GT3X-BT, Actigraph Inc.) on both the wrist and waist for 
the entire duration of the study to assess baseline physical activity and to account for any 
changes in physical activity during the trial. When/if limited availability of accelerometers arises, 
one accelerometer will be worn on the wrist. 
h4-day Diet Recall: Habitual Dietary Intake including average total calorie and macronutrient 
intake will be obtained prior to randomization from detailed 4-d diet records. Subjects will be 
instructed on procedures for weighing and recording food intake on 3 weekdays and one 
weekend day.  The ProNutra (version 3.5.0.1) will be used to calculate energy intake and 
macronutrient composition.   
Unscheduled Clinic Visit (2-5 days prior to Visit 2): 
• COVID-19 Nasal Swab to be completed prior to next visit. 
Visit 2 (Day -2) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Physical Activity Monitor Off 
Overnight Stay: Participants will be admitted to the Clinical Research Unit (CRU) in the evening 
and given a standard dinner of 7 kcal/kg consisting of 50% carbohydrate, 20% protein and 30% 
fat.  
Visit 2 (Day -1) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
iHyperinsulinemic Euglycemic Clamp: Following a 12-hour overnight fast at the CRU (including 
abstinence from caffeine containing foods and beverages), insulin sensitivity will be measured 
using the criterion method of the euglycemic clamp. Participants will have not performed 
vigorous physical activity for at least the previous 24 h, and be free of acute illness for the prior 2 
weeks. Subjects will undergo key laboratory visits at baseline and at prescribed time points 
during the dietary intervention. All female participants will be eumenorrheic and will begin the 
study during the early follicular phase (low estrogen and progesterone) of the menstrual cycle to 
minimize the impact of menstrual phase on substrate metabolism.  An intravenous catheter will 
be placed in an antecubital vein for subsequent insulin (40 mU/m2-min) and glucose infusions 
and for stable isotope (6, 6-2H2) infusions to measure insulin sensitivity. Insulin and FFA 
samples will be drawn at various time points throughout the clamp. 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 20 of 38 
Research Study protocol template version date 08/07/2020 
 
 
jRMR: Taken at baseline and last 30 minutes of the clamp using the AEI system. 
kMuscle Biopsy: One biopsy taken prior to clamp and a second biopsy taken 30-45 min after the 
start of the clamp. A biopsy of the Vastus Lateralis muscle will be performed on the leg/thigh 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 will be 
administered using 2% lidocaine. The skin will be incised (approximately 0.75 cm) with a #11 
scalpel, and the Bergstrom needle will be inserted into the Vastus Lateralis.  Approximately 220 
mg 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. 
Muscle specimens will be trimmed of adipose and connective tissues using a dissecting 
microscope, and analyzed as follows: 
• High-Resolution Respirometry. Mitochondrial respiratory capacity will be measured in 
permeabilized muscle fibers from vastus lateralis muscle biopsies (Oroboros Oxygraph 
2K, Innsbruck, Austria). The instrument has two chambers, which permits high-
resolution respiration measurements to be made in duplicate with low amounts of muscle 
tissue (2mg). The instrument employs a polarographic oxygen sensor with a high signal 
to noise ratio and accurate steady-state measurement of O2 levels and respiration with 
resolution > +/- 1 nmol O2 per liter. The O2K assay allows a comprehensive evaluation 
of mitochondrial respiratory function upon titration with ETC complex-specific inhibitors 
and substrates with both carbohydrate and fat as substrates (25, 26). 
• Protein and Gene Expression. Several proteins and genes related to insulin signaling 
(IRS, Akt, mTORC2, AS160, GLUT4) will be measured via Western blot quantification 
and Real-Time Quantitative PCR (RT-qPCR). Protein expression will be performed in 
duplicate in ~20 mg of the biopsy specimen obtained during both fasting and insulin-
stimulated conditions. Optimal antibody conditions for each protein have been 
established using aliquots from human muscle maintained in our lab for such purposes.  
• Immunohistochemistry. Histochemical analyses will be performed on serial sections using 
methods previously described (27). Histochemical analysis of IMCL (Oil Red O 
Staining), fiber-type and cross-sectional area will be measured in the fasted biopsy 
sample. In addition, LANCL2, GLUT4 and TXNIP translocation will be measured in 
sections from both the fasting and insulin stimulated biopsies (28).  
lBlood Draws: Baseline blood to be taken prior to the clamp and taken throughout the clamp at -
30, 0, 30, 60, 100, 110, and 120 min, plus every 10 min during the last 30 min of the clamp (220, 
230 and 240 min) for the GCMS determination of the [6, 6-2H2] glucose enrichment. Insulin and 
FFA samples will be drawn at various time points throughout the clamp. 
Randomization 
Post Procedure Lunch 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 21 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Visit 3 (Day 0) 
Vital Signs 
Weight 
Review AEs and Con Meds 
Biopsy Check 
Drug Distribution 
Drug Compliance 
Issuance of Stipend 
Visit 4 (Day 2), Visit 5 (Day 5), Visit 6 (Day 8), Visit 7 (Day 11) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Drug Distribution 
Drug Compliance 
Visit 8 (Day 13) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Physical Activity Monitor OFF 
Overnight Stay 
Visit 8 (Day 14) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Hyperinsulinemic Euglycemic Clamp 
RMR 
Muscle Biopsies 
Blood Draws 
Randomization 
Post Procedure Lunch 
Issuance of Stipend 
Visit 9 (Day 15) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 22 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Biopsy Check 
Drug Washout 
Visit 10 (Day 44) 
Vital Signs  
Weight 
Review AEs and Concomitant Medications 
DEXA 
Physical Activity Questionnaire 
Physical Activity Monitor On 
4-day Diet Recall 
 
Unscheduled Clinic Visit (2-5 days prior to next visit): 
• COVID-19 Nasal Swab to be completed prior to next visit. 
Visit 11 (Day 52) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Physical Activity Monitor Off 
Overnight Stay 
Visit 12 (Day 53) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Hyperinsulinemic Euglycemic Clamp 
RMR 
Muscle Biopsies 
Blood Draws 
Randomization 
Post Procedure Lunch 
Visit 12 (Day 54) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Biopsy Check 
Drug Distribution 
Drug Compliance 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 23 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Issuance of Stipend 
Visit 13 (Day 56), Visit 14 (Day 59), Visit 15 (Day 62), Visit 16 (Day 65) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Drug Distribution 
Drug Compliance 
 
Unscheduled Clinic Visit (2-5 days prior to next visit): 
• COVID-19 Nasal Swab to be completed prior to next visit. 
Visit 17 (Day 67) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Overnight Stay 
Visit 17 (Day 68) 
Vital Signs 
Weight 
Screening Blood Draw 
Urinalysis 
Review AEs and Concomitant Medications 
Hyperinsulinemic Euglycemic Clamp 
RMR 
Muscle Biopsies 
Blood Draws 
Physical Activity Monitor OFF 
Post Procedure Lunch 
Issuance of Stipend 
Visit 18 (Day 69) 
Vital Signs 
Weight 
Review AEs and Concomitant Medications 
Biopsy Check 
Measuring ABA in plasma.  Plasma will be isolated from blood samples collected from fasted 
subjects prior to the clamp and stored at –80 °C. Blood samples will be sent to BioTherapeutics 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 24 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Inc. after obtaining the Material Transfer Agreement, which will govern the transfer and chain of 
custody of the biospecimens outside of AH. Investigators at BioTherapeutics Inc. will measure 
ABAp as described (3), samples will be spiked with d-ABA (internal standard) in methanol, 
stored at –20 °C for 24 h, centrifuged for 10 min at 2,000 g and concentrated in a rotovaporator. 
The residue will be redissolved in DI water with the pH adjusted to ~2.5 with trifluoroacetic 
acid. Three extractions with diethyl ether will be performed; the ether layer will be collected and 
concentrated; the residue will be dissolved in DI water. Samples will be analyzed by LC/MS to 
quantify ABA levels. 
 
Study Duration 
 
All research visits will occur at the AdventHealth Translational Research Institute, 
Orlando. The project is planned to begin in October 2020, initiated by the recruitment of 
participants. For each participant undergoing the whole study protocol (including 
enrollment, following all the visits and related procedures), the total study duration is 
approximately 69 days (10 weeks). The estimated duration of the whole study completion 
including recruitment, clinical protocol, data collection, sample analyses, statistical 
analysis is about 2 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 the TRI Laboratory Room 2404. 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. 
 
Muscle Biopsy Specimens. Muscle specimens will be trimmed of any visible adipose 
and connective tissue using a dissecting microscope and processed and assays will be 
run as previously described. Any remaining tissue will be flash frozen in liquid nitrogen 
for future biochemical, molecular or genetic analyses. 
 
The muscle biopsy 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.  
 
Biospecimens collected will be stored at AdventHealth TRI until shipment to outside 
laboratories/institutions.  Laboratories that will analyze de-identified samples may 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 25 of 38 
Research Study protocol template version date 08/07/2020 
 
 
include but is not limited to BioTherapeutics Inc. and Quest Laboratory. When testing 
and analysis cannot be conducted at AdventHealth Orlando and will be conducted at 
outside laboratories, a Materials Transfer Agreement will be obtained.  The agreement 
will govern the transfer and chain of custody of the biospecimen to outside entities for 
testing and analysis. 
 
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 
scientists and scientists outside of AdventHealth.  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 Principal Investigator, with an additional review 
by the respective Program Director.  For research outside of AdventHealth, a Material 
Transfer Agreement will be obtained, which will govern the transfer and chain of 
custody of the biospecimens outside of AH. 
 
    
Study Outcome Measures (Endpoints) 
 
The primary outcome tolerability will be assessed via monitoring adverse events at each visit 
throughout the duration of the study. Additionally, before and immediately after the end of the 
trial the following examinations will be completed: complete physical exam, complete 
hemogram, comprehensive clinical chemistry profile, complete urine analysis. The second 
primary outcome (insulin sensitivity) will be measured using hyperinsulinic euglycemic clamp at 
visits 2, 8, 11, and 17.  
 
Secondary outcomes, including skeletal muscle biopsy-derived assessments and plasma-derived 
inflammatory, ABA and glucose control concentrations will be measured at visits 2, 8, 11, and 
17. 
 
 
Data Management and Quality Plan 
 
Data De-identification 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 26 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Participants will be enrolled using Cerner’s Patient Protocol Manager; the application 
assigns each participant a unique participant identifier, or “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 data is recorded in a de-identified 
fashion onto our paper source documents, which is then transcribed into our electronic case 
report forms, (CRF). The CRF is used for storage (a database) and facilitates analysis.  
Clinical data generated by research devices also uses the PID, and once the data has been 
transformed into interpretable results it is stored into the clinical research database. Both 
storage locations are secured and only assessable to the assigned clinical research team.  
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 clinical research database, where only the 
AdventHealth Translational Research Institute research team has access. These secure 
databases are stored/accessed on the AdventHealth password-protected computer network. 
No one outside of AdventHealth investigators or researchers will have access to the 
databases.         
     
Data Confidentiality, Storage, and Retention 
 
The identity and personal health information 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.  The data records will also be stored in as electronic records.  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.   
 
The duration of study data retention will be determined by governing FDA regulations 
and/or sponsor contract mandate (if applicable). 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. 
 
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. 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 27 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
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 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 (outside of AdventHealth Orlando)  
Sharing of anonymized data with other organizations would be allowed, as defined in 
respective Agreements, for research of any type if the research has scientific merit as 
determined by the TRI Scientific Review Committee. 
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.  
Should archived biospecimens be needed for research outside AdventHealth Orlando and 
certain data elements that are connected to the archived biospecimen samples are needed 
to conduct the research, then Data Use Agreement(s) will be obtained. The Data Use 
Agreement(s) will identify the purpose for data sharing, 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 
 
Power analyses has been conducted for repeated measures ANOVA. Based on published findings 
(3), we presume a minimum mean difference between the two treatments of 10.3 with a SD of 11.3 
and 11.9 at each of the data collection points. The correlation among the repeated measures is 
expected to be 0.8. Results of these simulations indicated that at alpha=0.05, a sample size of 24 
per treatment will achieve >80% power. We believe it is prudent to plan for some level of dropping 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 28 of 38 
Research Study protocol template version date 08/07/2020 
 
 
out. We will conservatively plan for a 20% dropout and recruit 30 total participants. We will 
continue to recruit and study subjects to achieve our desired sample size if dropouts exceed this 
estimate. 
 
Statistical Analysis Plan 
 
Analysis datasets will include: 
 
• Intention-to-Treat (ITT) Analysis Dataset (i.e., all randomized participants) 
• Modified Intention-to-Treat Analysis Dataset (e.g., participants who took at least one 
dose of study intervention and/or have some particular amount of follow-up outcome 
data) 
• Safety Analysis Dataset: defines the subset of participants for whom safety analyses will 
be conducted (e.g., participants who took at least one dose of study intervention) 
• Per-Protocol Analysis Dataset: defines a subset of the participants in the full analysis 
(ITT) set who complied with the protocol sufficiently to ensure that these data would be 
likely to represent the effects of study intervention according to the underlying scientific 
model (e.g., participants who took at least 80% of study intervention for 80% of the days 
within the maintenance period) 
• Other Datasets may be used for sensitivity analyses 
 
Prior to all analyses, the assumption of normality will be checked using histograms and 
normal probability plots. If normality is questionable, the nonparametric Wilcoxon rank 
sum test will be used. Multiple comparisons will be adjusted using Holm–Bonferroni 
method step down method.  
 
Primary Objective Analysis 
 
All analyses will be conducted using SAS or R package. We will begin by testing for any 
order effects in the randomized sequences (ABA vs. placebo) by using a two-sample t-test 
or non-parametric rank sum test (i.e. H0: μAB - μBA = 0). If the order effects are 
insignificant, data will be pooled from both arms to estimate treatment effects. We will use 
a multiple-sample repeated measures mixed model with a between-subject factors 
approach. Because the data are repeated, we will treat the multiple observations as nested 
within individuals. This will allow us to directly compare time points while accounting for 
the correlation in the data to make the correct inferences regarding differences between 
two conditions.  Here, with h = 1, .., s groups, i = 1, ….Nh subjects in group h and j = 1, 
…, n time points, the repeated measures model is: 𝛾𝛾ℎ𝑖𝑖𝑖𝑖=  𝜇𝜇+ 𝛾𝛾ℎ+ 𝜏𝜏𝑗𝑗 + (𝛾𝛾𝛾𝛾)ℎ𝑗𝑗+
 𝜋𝜋𝑖𝑖(ℎ) + 𝑒𝑒ℎ𝑖𝑖𝑖𝑖 ,  where 𝜇𝜇 = grand mean; 𝛾𝛾ℎ = effect of group h; 𝜏𝜏𝑗𝑗 = effect of time j; (𝛾𝛾𝛾𝛾)ℎ𝑗𝑗 
= interaction effect of time j and group h; 𝜋𝜋𝑖𝑖(ℎ) = individual difference component for 
subject i nested in group h, and 𝑒𝑒ℎ𝑖𝑖𝑖𝑖 = error for subject i in group h at time j. Confidence 
intervals and P-values will be adjusted for multiple comparisons of the individual effects.  

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 29 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Missing Data. We will use an intention-to-treat analysis as our primary analytic 
approach and rely on this analysis for interpreting the data. We will begin by examining 
the missing data patterns. Next, we will utilize maximum likelihood algorithms with the 
mixed linear model ANOVAs to longitudinally compare our outcomes across the three 
groups. Maximum likelihood algorithms estimations use all available data to construct 
weighted averages across the different patterns of missing data to provide valid point 
estimates and confidence intervals for population parameters as opposed to list-wise 
deletion. As a secondary analysis, we will conduct a completers-only analysis. However, 
in our experience both approaches yield similar conclusions. 
 
Secondary Objective Analysis 
Similar approaches will be uses as for primary endpoints. 
Potential Risks and Benefits 
 
Potential Benefits 
 
Importantly, the proposed project might have direct, tangible benefits for participants. 
These benefits include information about their health. All study participants will be 
encouraged to communicate the results from the study to their primary care providers. 
 
Potential Risks 
 
 
Potential risks are those associated with health information privacy, venipuncture, ABA 
supplementation and radiation from DEXA.  
Blood draws: There is a risk of pain, vasovagal syncope, hematomas, and/or infection at 
IV insertion/blood draw site (low risk of serious AEs).   
ABA Supplementation: The risks presented to subjects are minimal, and information 
gained from this project will be useful in assessing how metabolism in prediabetes and 
T2D is influenced by nutrition. Abscisic acid is endogenously produced and consumed 
within the standard diet. Studies assessing the presence of abscisic acid within blood and 
tissue have identified nanogram amounts. We have determined ABA supplementation is 
generally recognized as safe (GRAS) in accordance with FDA guidance. The matching 
placebo supplementation is made using corn products, which could pose a mild risk to 
those participants that experience a corn allergy. Participants will be screened for corn 
allergies and excluded if appropriate. 
DEXA Scan: 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. 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 30 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
Muscle 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. 
 
RMR (Resting Metabolic Rate): The measure of your metabolic rate using a ventilated 
hood carries no risk.  The only adverse factor about this testing may be a feeling of 
claustrophobia.  A member of the study staff will be at the bedside at all times and will 
check to see that you are comfortable.  The transparent hood can easily be removed, if 
necessary 
Activity Monitor:  There are no anticipated risks with the activity monitor itself, 
however it is possible you may experience some minor skin irritation from the monitor 
strap.  If you have allergies to metal (especially nickel) you will not be required to wear 
the monitor. 
Glucose clamp: Minor risks of this procedure include bruising at the site of the needle 
insertion in your veins.  The major risk of this procedure is low blood sugar due to the 
insulin given.  Low blood sugar can make you feel nauseous, sweaty, irritable and 
sometimes confused.  To prevent this, your blood sugar will be checked every 5-10 
minutes, and you will be given glucose through the IV line if needed. 
 
Mitigation of Risks  
 
Blood draws (lab samples, e.g.). There is a risk of pain, vasovagal syncope, hematomas, 
and/or infection blood draw site (low risk of serious AEs).  
Protection Against Risk: 
All venipuncture will be conducted by qualified staff using aseptic techniques. Total blood 
volume throughout the entire study will be approximately 110 mls.  
 
DEXA scan. 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. 
  
Protection Against Risk: 
             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). 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 31 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Muscle 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.  
 
Protection Against Risk: 
            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 to lidocaine and 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. 
 
RMR (Resting Metabolic Rate): The measure of your metabolic rate using a ventilated 
hood carries no risk.  The only adverse factor about this testing may be a feeling of 
claustrophobia.  A member of the study staff will be at the bedside at all times and will 
check to see that you are comfortable.  The transparent hood can easily be removed, if 
necessary 
 
Protection Against Risk: 
A member of the study staff will be at the bedside at all times and will check to see that 
you are comfortable.  The transparent hood can easily be removed, if necessary 
 
Activity Monitor:  There are no anticipated risks with the activity monitor itself, however 
it is possible you may experience some minor skin irritation from the monitor strap.  If you 
have allergies to metal (especially nickel) you will not be required to wear the monitor. 
 
Glucose clamp: Minor risks of this procedure include bruising at the site of the needle 
insertion in your veins.  The major risk of this procedure is low blood sugar due to the 
insulin given.  Low blood sugar can make you feel nauseous, sweaty, irritable and 
sometimes confused.  To prevent this, your blood sugar will be checked every 5-10 
minutes, and you will be given glucose through the IV line if needed. 
 
Protection Against Risk: 
To prevent this, your blood sugar will be checked every 5-10 minutes, and you will be 
given glucose through the IV line if needed. 
 
Again, written informed consent will be obtained after explanation to subjects about all 
procedures and time commitments. The study interviewers will explain to prospective 
participants the purpose, methods and extent of the study. Potential participants will be 
asked to read the informed consent form and to ask questions. The form will be written 
in simple, easy-to-understand language. Staff members will also review all key aspects 
of the study verbally. Confidentiality of data will be maintained by using research 
identification numbers that uniquely identify each individual. Data will be used only in 
aggregate and no identifying characteristics of individuals will be published or 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 32 of 38 
Research Study protocol template version date 08/07/2020 
 
 
presented. Finally, the study protocol has been registered at www.ClinicalTrials.gov 
(NCT#03295734) and will be updated as required. 
 
Provisions to Protect the Privacy Interest of Subjects 
 
Subjects will be assigned unique identifiers for study-related records. All precautions will 
be taken to make sure that only authorized individuals will access subject research records. 
Consenting, eligibility criteria, research intervention and procedures are conducted in a 
private room.  The collection of personal and sensitive information is limited to only 
necessary information. Private health information will be collected, accessed, and stored 
according to HIPAA guidelines. Data on subjects will be kept in locked areas and/or in 
secure research databases to ensure confidentiality. Blood and tissue specimens, when 
applicable, will be bar coded in research storage freezers. Code numbers will be assigned 
to patients for data entry purposes, and the data will be kept on a password protected server. 
 
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; or 
• Administrative reasons require the participant’s withdrawal. 
 
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 a participant may have 
withdrawn due to possible drug side effects (if applicable) and keeping these participants 
in the analysis is essential for study validity. 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 33 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
Adverse Event Reporting 
 
An adverse event (AE) is defined as both an expected side effect that is of a serious nature, or an 
unexpected side effect/event regardless of severity. Each participant is 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 FH 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 will be recorded in the source document. All adverse and unexpected events 
will be reported according to AdventHealth IRB guidelines. 
Adverse events will be documented and reported by the study coordinator, Medical Investigator 
or other TRI 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. All adverse events and unexpected 
and/or severe adverse events will be reported to the AdventHealth Orlando IRB. The TRI has a 
standing committee that meets monthly to review all adverse events in our clinical trials and will 
additionally be charged with review of the study 
All adverse events will be reported according to AdventHealth IRB guidelines. 
 
Safety Monitoring Plan   
 
Safety Monitoring 
 
Safety of participants is our primary concern. Prior to study entry, prospective participants 
will be advised to consult with their physician. Numerous safety procedures will be utilized 
to ensure participant safety. 
 
First, many of the exclusion criteria are designed to exclude those at moderate to high risk. 
Participant education about safety begins with the consent process and continues 
throughout the study. Potential adverse events for study related activities and interventions 
are explained to each participant by trained study personnel during the informed consent 
process. Participants will be encouraged to notify study staff immediately if they have any 
adverse experiences that could be related to the study drug. Abnormal symptoms or vital 
signs detected during intervention visits will be discussed with the study physician in real 
time, who will dictate the appropriate course of action. 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 34 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
Center-based assessments and interventions will be conducted and supervised by trained 
staff who will monitor potential adverse experiences and symptoms. All assessors and 
interventionists receive CPR training and training on management of acute events 
including syncope, chest pain, acute dyspnea, focal neurological symptoms and abnormal 
vital signs. Portable defibrillators are available, and all study staff have on-call access to 
the study physician and contact numbers for emergency services are. Institutional and 
community EMS services will be activated if needed. 
 
Any significant adverse events will be reported promptly to the IRB and to the Data 
Safety Monitoring Board (DSMB). Again, written informed consent will be obtained 
after explanation to subjects about all procedures and time commitments. The study 
interviewers will explain to prospective participants the purpose, methods and extent of 
the study. Potential participants will be asked to read the informed consent form and to 
ask questions. The form will be written in simple, easy-to-understand language. Staff 
members will also review all key aspects of the study verbally. Confidentiality of data 
will be maintained by using research identification numbers that uniquely identify each 
individual. Data will be used only in aggregate and no identifying characteristics of 
individuals will be published or presented. 
 
The muscle biopsy procedure will be performed by a trained and experienced study 
healthcare provider. The muscle biopsy procedure will be performed under sterile 
conditions using aseptic techniques. Local anesthesia is obtained using subcutaneous 
lidocaine. The incision will be closed with a sterile suture and covered with a pressure 
dressing. The risk of the participants experiencing an allergic reaction is this injection is 
extremely low, and to reduce the risk of any potential adverse reactions occurring, 
participants will be queried prior to the procedure to verify that they do not have a known 
allergy to lidocaine.   
 
Data and Safety Monitoring Board (DSMB) or Equivalent 
 
Among the most comprehensive and effective method of monitoring risks in studies with 
relatively few subjects is a weekly and biweekly individual case that will be reviewed by 
the PI, one of the study physicians, and/or research coordinator including progress or 
adverse events occurring in the following: subject confidentiality, subject recruitment, 
and consent process. All will monitor response to tolerance of and effectiveness of the 
exercise program. Files will be kept in a locked file cabinet in the laboratory of the PI to 
assure individual privacy and confidentiality of subject records. The research 
coordinators will monitor the following items: the timeframe of recruiting subjects, 
quality of data being entered, any external factors relevant for the safety of participants 
throughout the entire study, and is also instructed to make the PI aware of all events, 
expected or unexpected. 
 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 35 of 38 
Research Study protocol template version date 08/07/2020 
 
 
Ethical Considerations 
 
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.  
No other specific ethical considerations are identified for this study.  
 
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 when appropriate and approved by the PI. 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 is supported by funding from the National Institute of Health. 
 
Subject Stipends or Payments 
 
Participants will receive a total of $1200 upon completion of all study visits. If a participant is 
unable to complete all study visits, the stipends will be prorated to the following: 
 
Visit 
Prorated Amount 
After completion of V2 
$400 
After completion of V8 
$400 
After completion of V18 
$400 
 
A   Payment Card will be the method of payment and will be requested upon completion of the study 
or on their last day of participation, should they find they cannot complete the study.   
 
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). The goal will be to publish novel and interesting findings 
from this research. 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. 
   
 
 
 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 36 of 38 
Research Study protocol template version date 08/07/2020 
 
 
 
References 
1. 
Bruzzone S, Magnone M, Mannino E, Sociali G, Sturla L, Fresia C, Booz V, Emionite L, De Flora A, 
Zocchi E. Abscisic Acid Stimulates Glucagon-Like Peptide-1 Secretion from L-Cells and Its Oral Administration 
Increases Plasma Glucagon-Like Peptide-1 Levels in Rats. PLoS One. 2015;10(10):e0140588. doi: 
10.1371/journal.pone.0140588. PubMed PMID: 26488296; PMCID: PMC4619318. 
2. 
Bruzzone S, Bodrato N, Usai C, Guida L, Moreschi I, Nano R, Antonioli B, Fruscione F, Magnone M, 
Scarfi S, De Flora A, Zocchi E. Abscisic acid is an endogenous stimulator of insulin release from human pancreatic 
islets with cyclic ADP ribose as second messenger. J Biol Chem. 2008;283(47):32188-97. doi: 
10.1074/jbc.M802603200. PubMed PMID: 18784081. 
3. 
Magnone M, Ameri P, Salis A, Andraghetti G, Emionite L, Murialdo G, De Flora A, Zocchi E. Microgram 
amounts of abscisic acid in fruit extracts improve glucose tolerance and reduce insulinemia in rats and in humans. 
FASEB J. 2015;29(12):4783-93. doi: 10.1096/fj.15-277731. PubMed PMID: 26243865. 
4. 
Ameri P, Bruzzone S, Mannino E, Sociali G, Andraghetti G, Salis A, Ponta ML, Briatore L, Adami GF, 
Ferraiolo A, Venturini PL, Maggi D, Cordera R, Murialdo G, Zocchi E. Impaired increase of plasma abscisic Acid 
in response to oral glucose load in type 2 diabetes and in gestational diabetes. PLoS One. 2015;10(2):e0115992. doi: 
10.1371/journal.pone.0115992. PubMed PMID: 25723556; PMCID: PMC4344322. 
5. 
Zocchi E, Hontecillas R, Leber A, Einerhand A, Carbo A, Bruzzone S, Tubau-Juni N, Philipson N, 
Zoccoli-Rodriguez V, Sturla L, Bassaganya-Riera J. Abscisic Acid: A Novel Nutraceutical for Glycemic Control. 
Front Nutr. 2017;4:24. doi: 10.3389/fnut.2017.00024. PubMed PMID: 28660193; PMCID: PMC5468461. 
6. 
Bruzzone S, Ameri P, Briatore L, Mannino E, Basile G, Andraghetti G, Grozio A, Magnone M, Guida L, 
Scarfi S, Salis A, Damonte G, Sturla L, Nencioni A, Fenoglio D, Fiory F, Miele C, Beguinot F, Ruvolo V, Bormioli 
M, Colombo G, Maggi D, Murialdo G, Cordera R, De Flora A, Zocchi E. The plant hormone abscisic acid increases 
in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts. FASEB J. 
2012;26(3):1251-60. doi: 10.1096/fj.11-190140. PubMed PMID: 22075645. 
7. 
Rehm CD, Penalvo JL, Afshin A, Mozaffarian D. Dietary Intake Among US Adults, 1999-2012. JAMA. 
2016;315(23):2542-53. doi: 10.1001/jama.2016.7491. PubMed PMID: 27327801. 
8. 
Lu P, Bevan DR, Lewis SN, Hontecillas R, Bassaganya-Riera J. Molecular modeling of lanthionine 
synthetase component C-like protein 2: a potential target for the discovery of novel type 2 diabetes prophylactics 
and therapeutics. J Mol Model. 2011;17(3):543-53. doi: 10.1007/s00894-010-0748-y. PubMed PMID: 20512604. 
9. 
Sturla L, Fresia C, Guida L, Grozio A, Vigliarolo T, Mannino E, Millo E, Bagnasco L, Bruzzone S, De 
Flora A, Zocchi E. Binding of abscisic acid to human LANCL2. Biochemical and biophysical research 
communications. 2011;415(2):390-5. doi: 10.1016/j.bbrc.2011.10.079. PubMed PMID: 22037458. 
10. 
Sturla L, Fresia C, Guida L, Bruzzone S, Scarfi S, Usai C, Fruscione F, Magnone M, Millo E, Basile G, 
Grozio A, Jacchetti E, Allegretti M, De Flora A, Zocchi E. LANCL2 is necessary for abscisic acid binding and 
signaling in human granulocytes and in rat insulinoma cells. J Biol Chem. 2009;284(41):28045-57. doi: 
10.1074/jbc.M109.035329. PubMed PMID: 19667068; PMCID: PMC2788856. 
11. 
Zeng M, van der Donk WA, Chen J. Lanthionine synthetase C-like protein 2 (LanCL2) is a novel regulator 
of Akt. Molecular biology of the cell. 2014;25(24):3954-61. doi: 10.1091/mbc.E14-01-0004. PubMed PMID: 
25273559; PMCID: 4244203. 
12. 
Sato M, Dehvari N, Oberg AI, Dallner OS, Sandstrom AL, Olsen JM, Csikasz RI, Summers RJ, 
Hutchinson DS, Bengtsson T. Improving type 2 diabetes through a distinct adrenergic signaling pathway involving 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 37 of 38 
Research Study protocol template version date 08/07/2020 
 
 
mTORC2 that mediates glucose uptake in skeletal muscle. Diabetes. 2014;63(12):4115-29. doi: 10.2337/db13-1860. 
PubMed PMID: 25008179. 
13. 
Lu P, Hontecillas R, Horne WT, Carbo A, Viladomiu M, Pedragosa M, Bevan DR, Lewis SN, Bassaganya-
Riera J. Computational modeling-based discovery of novel classes of anti-inflammatory drugs that target lanthionine 
synthetase C-like protein 2. PLoS One. 2012;7(4):e34643. doi: 10.1371/journal.pone.0034643. PubMed PMID: 
22509338; PMCID: 3324509. 
14. 
Bassaganya-Riera J, Guri AJ, Lu P, Climent M, Carbo A, Sobral BW, Horne WT, Lewis SN, Bevan DR, 
Hontecillas R. Abscisic acid regulates inflammation via ligand-binding domain-independent activation of 
peroxisome proliferator-activated receptor gamma. J Biol Chem. 2011;286(4):2504-16. doi: 
10.1074/jbc.M110.160077. PubMed PMID: 21088297; PMCID: 3024745. 
15. 
Guri AJ, Hontecillas R, Si H, Liu D, Bassaganya-Riera J. Dietary abscisic acid ameliorates glucose 
tolerance and obesity-related inflammation in db/db mice fed high-fat diets. Clin Nutr. 2007;26(1):107-16. doi: 
10.1016/j.clnu.2006.07.008. PubMed PMID: 17000034. 
16. 
Guri AJ, Hontecillas R, Ferrer G, Casagran O, Wankhade U, Noble AM, Eizirik DL, Ortis F, Cnop M, Liu 
D, Si H, Bassaganya-Riera J. Loss of PPAR gamma in immune cells impairs the ability of abscisic acid to improve 
insulin sensitivity by suppressing monocyte chemoattractant protein-1 expression and macrophage infiltration into 
white adipose tissue. J Nutr Biochem. 2008;19(4):216-28. doi: 10.1016/j.jnutbio.2007.02.010. PubMed PMID: 
17618105. 
17. 
Magnone M, Ameri P, Salis A, Andraghetti G, Emionite L, Murialdo G, De Flora A, Zocchi E. Microgram 
amounts of abscisic acid in fruit extracts improve glucose tolerance and reduce insulinemia in rats and in humans. 
FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2015. doi: 
10.1096/fj.15-277731. PubMed PMID: 26243865. 
18. 
Kato-Noguchi H, Tanaka Y, Murakami T, Yamamura S, Fujihara S. Isolation and identification of an 
allelopathic substance from peel of Citrus junos. Phytochemistry. 2002;61(7):849-53. PubMed PMID: 12453578. 
19. 
Le Page-Degivry MT, Bidard JN, Rouvier E, Bulard C, Lazdunski M. Presence of abscisic acid, a 
phytohormone, in the mammalian brain. Proc Natl Acad Sci U S A. 1986;83(4):1155-8. PubMed PMID: 2937056; 
PMCID: PMC323030. 
20. 
Amati F, Dube JJ, Alvarez-Carnero E, Edreira MM, Chomentowski P, Coen PM, Switzer GE, Bickel PE, 
Stefanovic-Racic M, Toledo FG, Goodpaster BH. Skeletal muscle triglycerides, diacylglycerols, and ceramides in 
insulin resistance: another paradox in endurance-trained athletes? Diabetes. 2011;60(10):2588-97. doi: 
10.2337/db10-1221. PubMed PMID: 21873552; PMCID: PMC3178290. 
21. 
Coen PM, Dube JJ, Amati F, Stefanovic-Racic M, Ferrell RE, Toledo FG, Goodpaster BH. Insulin 
resistance is associated with higher intramyocellular triglycerides in type I but not type II myocytes concomitant 
with higher ceramide content. Diabetes. 2010;59(1):80-8. doi: 10.2337/db09-0988. PubMed PMID: 19833891; 
PMCID: 2797948. 
22. 
Coen PM, Hames KC, Leachman EM, DeLany JP, Ritov VB, Menshikova EV, Dube JJ, Stefanovic-Racic 
M, Toledo FG, Goodpaster BH. Reduced skeletal muscle oxidative capacity and elevated ceramide but not 
diacylglycerol content in severe obesity. Obesity (Silver Spring). 2013;21(11):2362-71. doi: 10.1002/oby.20381. 
PubMed PMID: 23512750; PMCID: PMC4136513. 
23. 
Coen PM, Menshikova EV, Distefano G, Zheng D, Tanner CJ, Standley RA, Helbling NL, Dubis GS, 
Ritov VB, Xie H, Desimone ME, Smith SR, Stefanovic-Racic M, Toledo FG, Houmard JA, Goodpaster BH. 
Exercise and Weight Loss Improve Muscle Mitochondrial Respiration, Lipid Partitioning, and Insulin Sensitivity 

 
Current Version Date: 
02.09.2023 
IRBNet #: 
1545505 
  
 
Page 38 of 38 
Research Study protocol template version date 08/07/2020 
 
 
After Gastric Bypass Surgery. Diabetes. 2015;64(11):3737-50. doi: 10.2337/db15-0809. PubMed PMID: 26293505; 
PMCID: PMC4613980. 
24. 
Dube JJ, Coen PM, DiStefano G, Chacon AC, Helbling NL, Desimone ME, Stafanovic-Racic M, Hames 
KC, Despines AA, Toledo FG, Goodpaster BH. Effects of acute lipid overload on skeletal muscle insulin resistance, 
metabolic flexibility, and mitochondrial performance. Am J Physiol Endocrinol Metab. 2014;307(12):E1117-24. 
doi: 10.1152/ajpendo.00257.2014. PubMed PMID: 25352435; PMCID: 4269675. 
25. 
Hutter E, Unterluggauer H, Garedew A, Jansen-Durr P, Gnaiger E. High-resolution respirometry--a modern 
tool in aging research. Experimental gerontology. 2006;41(1):103-9. Epub 2005/11/29. doi: 
10.1016/j.exger.2005.09.011. PubMed PMID: 16309877. 
26. 
Gnaiger E. Capacity of oxidative phosphorylation in human skeletal muscle: new perspectives of 
mitochondrial physiology. The international journal of biochemistry & cell biology. 2009;41(10):1837-45. Epub 
2009/05/27. doi: 10.1016/j.biocel.2009.03.013. PubMed PMID: 19467914. 
27. 
Behan WM, Cossar DW, Madden HA, McKay IC. Validation of a simple, rapid, and economical technique 
for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle. Journal of clinical pathology. 
2002;55(5):375-80. Epub 2002/05/03. PubMed PMID: 11986345; PMCID: Pmc1769641. 
28. 
Bradley H, Shaw CS, Worthington PL, Shepherd SO, Cocks M, Wagenmakers AJ. Quantitative 
immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance 
and sprint interval training. Physiological reports. 2014;2(7). Epub 2014/07/24. doi: 10.14814/phy2.12085. PubMed 
PMID: 25052490; PMCID: PMC4187550.