Clinical trial NCT02844907
Conditions: Insulin Secretion
Keywords: Incretins GLP-1 Exendin-9 Insulin Resistance Diabetes Pre-diabetes
Sponsor: VA Office of Research and Development
Start date: 2018-07-01; Completion date: 2021-12-31
Enrollment: 6 participants; Allocation: NON_RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
AIM2: The purpose of Aim-2 of this study is to determine the role of basal GLP-1 action on the beta-cell response to insulin resistance. Healthy subjects will have fasting GLP-1 action determined with GLP-1r blockade before and after induction of experimental insulin resistance. The investigators hypothesize that fasting GLP-1 action will increase to compensate for experimental insulin resistance.

AIM3: The purpose of Aim-3 of this study is to determine the role of basal GLP-1 action on fasting glucose regulation in lean, obese, pre-diabetic and type 2 diabetic (T2DM) subjects. A cross sectional study of age-matched subjects across the spectrum of glucose tolerance will be used to test the hypothesis that fasting GLP-1 action increases as beta-cell function declines.

Detailed description:
AIM2: The purpose of this study is to determine the role of basal GLP-1 action on the beta-cell response to insulin resistance. Healthy subjects will have fasting GLP-1 action determined with GLP-1r blockade before and after induction of experimental insulin resistance. The investigators hypothesize that fasting GLP-1 action will increase to compensate for experimental insulin resistance.

The study will enroll up to 20-25 healthy participants and while it is anticipated that the screening blood draw will yield a number of screen failures, it is estimated that 10-15 subjects will complete the entire study protocol. Enrolled participants will be asked to complete three study visits including the consent visit and two infusion study visits that will include hyperglycemic clamp procedures with the addition of the GLP-1 receptor antagonist Exendin (9-39) to determine the fasting GLP-1 effect. Each infusion procedure will last 2 hours. Subjects will be asked to take dexamethasone daily for approximately one week between Visit-2 and Visit-3 to induce insulin resistance.

The investigators will enroll 6-10 additional subjects as controls for effects of time and repeat testing. These individuals will have identical clamps with no dexamethasone at approximately 1 week intervals and will be determined at random by study staff.

The primary outcome for visits 2-3 will be to measure the effects of Ex-9 on fasting glucose-stimulated insulin secretion before and after experimentally induced insulin resistance. The primary experimental variable for analysis will be C-peptide during the clamp. Mean values will be compared between the period of glucose only stimulation and glucose with Ex-9. For each subject in the active treatment arm data will be analyzed using 2-way ANOVA for repeated measures using time (0-60 vs 60-120 min) and treatment (Dex vs no Dex) as the two factors. Based on the investigators' previous studies the investigators expect a significant time effect due to Ex-9. If there is an interaction with treatment the investigators would conclude that experimental insulin resistance influences the fasting GLP-1 effect. Data from control subjects will be analyzed identically; here the investigators expect no interaction, indicating that the fasting GLP-1 is a stable measure. In the investigators' previous study using Ex-9 during a glucose clamp, the average coefficient of variation in insulin concentrations at the conclusion of each step in the ramp was 30%. Using this estimate of between subject variation, detecting a 20% difference between subsequent steps in the GLP-1 ramp with a power of 80% and significance level of 0.05 will require 8 subjects.

AIM3: The purpose of Aim-3 of this study is to determine the role of basal GLP-1 action on fasting glucose regulation in lean, obese, pre-diabetic and type 2 diabetic (T2DM) subjects. Approximately 15-20 subjects will complete this cross sectional study of age-matched subjects across the spectrum of glucose tolerance will be used to test the hypothesis that fasting GLP-1 action increases as beta-cell function declines.

Enrolled participants for Aim-3 will be asked to complete two study visits including the consent visit and one infusion visit that will include a hyperglycemic clamp and an infusion of the antagonist Exendin (9-39). The infusion procedure will last approximately 2 hours.

The primary outcome measure for Aim-3 will be the fasting GLP-1 effect. This will be defined as the difference in steady state glucose-stimulated insulin secretion with and without Ex-9. The difference will be expressed as a percentage of glucose stimulated insulin secretion without Ex-9, and serve as the primary variable for comparison among the lean, obese and diabetic cohorts.

Arms: arm 1: During the 2-hour procedure, a variable infusion of 20% dextrose and blood glucose will be clamped at basal + 3mM. Participants will receive an infusion of the GLP-1 receptor antagonist Exendin (9-39) between the 60-120 minute time-points of the clamp. The amount of Ex-9 infused will be 750 pmol/kg/min for 60 minutes. arm 2: Subjects will be asked to take 4 mg once daily between Visit-2 and Visit-3.
Interventions: intervention 1: Exendin (9-39) intervention 2: Dexamethasone intervention 3: Hyperglycemic Clamp
Intervention descriptions: intervention 1: Upon establishing a hyperglycemic clamp (target blood glucose at basal +3 mM) Exendin (9-39) will be infused. intervention 2: Between Visit-2 and Visit-3, Dexamethasone (4 mg tablet) once daily for 5-7 days. intervention 3: A variable infusion of 20% dextrose will begin to clamp the blood glucose at basal +3 mM.

Protocol documents:
Prot_SAP_000.pdf: 
Study Title: Regulation of Insulin Secretion by the GLP-1 Receptor 
Principal Investigator: David D'Alessio 
NCT02844907 
Document Approval Date: 06/18/2020 


Research Abstract
Please type your Research Abstract here:
The Research Abstract should summarize the main points of your study in one paragraph. The following 
guidelines may help you:
1. Purpose and objective (1-2 sentences)
2. Study activities and population group (2-4 sentences)
3. Data analysis and risk/safety issues (1-2 sentences)
This protocol is funded by the Veteran’s Administration and being led by Dr. David D’Alessio in the 
Division of Endocrinology and the Duke Molecular Physiology Institute.
PURPOSE OF STUDY:
1. Determine the interaction of GLP-1 and GIP to regulate insulin secretion in nondiabetic subjects.
The first part of this aim will determine the threshold plasma concentration of GLP-1 that increases
insulin secretion to test the hypothesis that fasting levels of circulating GLP-1 are insufficient to
mediate β-cell secretion. The second part of the aim will determine whether the insulin response to
specific amino acids is dependent on the α-cell release of GLP-1.
2. Determine the role of basal GLP-1 action on the β-cell response to insulin resistance. Healthy
subjects will have fasting GLP-1 action determined with GLP-1r blockade before and after induction of
experimental insulin resistance. We hypothesize that fasting GLP-1 action will increase to compensate
for experimental insulin resistance.
3. Determine the role of basal GLP-1 action on fasting glucose regulation in lean, obese, pre-diabetic
and diabetic subjects. A cross sectional study of age-matched subjects across the spectrum of glucose
tolerance will be used to test the hypothesis that fasting GLP-1 action increases as β-cell function
declines. Groups of lean, obese, pre-diabetic and type 2 diabetic (T2DM) subjects will receive exedin-9
during a graded glucose infusion. We hypothesize that the relative effect of GLP-1r blockade on insulin
secretion will be greater with progression from normal to T2DM.
The initial protocol submitted and approved by the Duke IRB was intended to focus solely on the 
design and procedures for Aim 1 in order to address the three aims listed below in sequential order 
such that the findings from Aim 1 would be used to further develop the protocol design and 
procedures for Aim 2, and similarly the findings from Aims 1 and 2 will strengthen the approach to 
Aim 3. 
This study will enroll up to 40-60 total participants 
While we anticipate that the 
for Aim-2 and Aim-3. 
screening blood draw will yield a number of screen failures, we expect that approximately 20-25 
subjects will complete Aim-2 and 15-20 will complete Aim-3 of the study protocol. 
Enrolled participants for Aim-2 will be asked to complete three study visits including the consent visit 
and two infusion study visits that will include hyperglycemic clamps and an infusion of 
. Each 
Exedin-9
DUHS IRB Application (Version 1.5)
General Information
*Please enter the full title of your protocol:
Regulation of Insulin Secretion by the GLP-1 Receptor
*Please enter the Short Title you would like to use to reference the study:
Paracrine Effects on Insulin Secretion
* This field allows you to enter an abbreviated version of the Study Title to quickly identify this
study.


infusion procedure will last 2 hours.
AIM-2: The primary outcome for visits 2-3 will be to measure the effects of Ex-9 on fasting glucose-
stimulated insulin secretion before and after experimentally induced insulin resistance. The primary 
experimental variable for analysis will be C-peptide during the clamp. Mean values will be compared 
between the period of glucose only stimulation and glucose with Ex-9. For each subject in the active 
treatment arm data will be analyzed using 2-way ANOVA for repeated measures using time (0-60 vs 
60-120 min) and treatment (Dex vs no Dex) as the two factors. Based on our previous studies we
expect a significant time effect due to Ex-9. If there is an interaction with treatment we would
conclude that experimental insulin resistance influences the fasting GLP-1 effect. Data from control
subjects will be analyzed identically; here we expect no interaction, indicating that the fasting GLP-1
is a stable measure. In our previous study using Ex-9 during a glucose clamp, the average coefficient
of variation in insulin concentrations at the conclusion of each step in the ramp was 30%. Using this
estimate of between subject variation, detecting a 20% difference between subsequent steps in the
GLP-1 ramp with a power of 80% and significance level of 0.05 will require 8 subjects.
Enrolled participants for Aim-3 will be asked to complete two study visits including the consent visit 
and one infusion study visit that will include a hyperglycemic clamp 
. The 
and an infusion of Exedin-9
infusion procedure will last 2 hours.
AIM-3 – The primary outcome measure for this study will be the fasting GLP-1 effect. This will be 
defined as the difference in steady state glucose-stimulated insulin secretion with and without Ex-9. 
The difference will be expressed as a percentage of glucose stimulated insulin secretion without Ex-9, 
and serve as the primary variable for comparison among the lean, obese and diabetes cohorts. 
Research Summary
State your primary study objectives
AIM-2: Determine the role of basal GLP-1 action on the β-cell response to insulin resistance.  Healthy 
subjects will have fasting GLP-1 action determined with GLP-1r blockade before and after induction of 
experimental insulin resistance.  We hypothesize that fasting GLP-1 action will increase to compensate for 
experimental insulin resistance.
AIM-3: The primary outcome measure for this study will be the fasting GLP-1 effect. This will be defined as 
the difference in steady state glucose-stimulated insulin secretion with and without Ex-9. 
State your secondary study objectives
Please select your research summary form:
Standard Research Summary Template
This is the regular (generic) research summary template which is required  for all regular applications (unless 
your protocol fits under the other research summary templates in this category).  Use of these instructions is 
helpful for ensuring that the research summary contains all necessary elements.
Standard Research Summary
Purpose of the Study
Objectives & hypotheses to be tested


1.  
2.  
3.  
This protocol is funded by the Veteran’s Administration and being led by Dr. David D’Alessio in the Division 
of Endocrinology and the Duke Molecular Physiology Institute. The study will address the following three 
specific aims:
Determine the roles of fasting GLP-1 concentrations and alpha-cell secretion on insulin 
release.  The first part of this aim will determine the threshold plasma concentration of GLP-1 that 
increases insulin secretion to test the hypothesis that fasting levels of circulating GLP-1 are 
insufficient to mediate β-cell secretion.  The second part of the aim will determine whether the 
insulin response to specific amino acids is dependent on the α-cell release of GLP-1.
Determine the role of basal GLP-1 action on the β-cell response to insulin resistance.  Heal
thy subjects will have fasting GLP-1 action determined with GLP-1r blockade before and after 
induction of experimental insulin resistance.  We hypothesize that fasting GLP-1 action will increase 
to compensate for experimental insulin resistance.
Determine the role of basal GLP-1 action on fasting glucose regulation in lean, obese, pre-
diabetic and diabetic subjects.  A cross sectional study of age-matched subjects across the 
spectrum of glucose tolerance will be used to test the hypothesis that fasting GLP-1 action 
increases as β-cell function declines.  Groups of lean, obese, pre-diabetic and type 2 diabetic 
(T2DM) subjects will receive exedin-9 during a graded glucose infusion.  We hypothesize that the 
relative effect of GLP-1r blockade on insulin secretion will be greater with progression from normal 
to T2DM.
Initially, this study was to address these three aims in sequential order such that the findings from Aim 1 
would be used to further develop the protocol design and procedures for Aim 2, and similarly the findings 
from Aims 1 and 2 will strengthen the approach to Aim 3. To that end, Dr. D’Alessio has been able to 
address Aim 1 by collecting and analyzing data from another study with a similar protocol design.  This IRB 
amendment is being filed to address the procedures for both Aim 2 and Aim 3.
Background & Significance
Should support the scientific aims of the research
GLP-1 is a potent insulin secretagogue that has a critical role in the incretin effect. The 
conventional model of GLP-1 action is that it acts as a hormone, released from enteroendocrine cells into 
the circulation during nutrient absorption, mediating its effects directly on target tissues like the b-cell. 
However, this model has been questioned because of the relatively small dynamic range of postprandial 
GLP-1 secretion, and its rapid inactivation by DPP-4. We have recently reported that in mice GLP-1 
signaling is necessary for normal glucose tolerance, but this effect is independent of circulating GLP-1. 
Moreover, in studies of healthy and diabetic humans we have observed that blockade of the GLP-1 
receptor (GLP-1r) reduces glucose-stimulated insulin secretion in the fasting state when plasma active GLP-
1 levels are low and unchanging. These findings are consistent with an emerging body of work questioning 
an endocrine model of GLP-1 action. One hypothesis advanced as an alternative is that GLP-1 produced in 
islet a-cells acts in a paracrine manner to regulate neighboring b-cells. This model is supported by several 
recent reports demonstrating islet production and action of GLP-1. In fact, evidence from rodent studies, 
and from our work in humans, indicates that there is a greater effect of islet GLP-1 signaling during 
periods of increased b-cell demand. Taken together, the published literature raises the possibility that intra-
islet GLP-1 action is a mechanism of b-cell compensation. If true, this model would dramatically shift 
understanding of the incretin system and present a range of new opportunities for diabetes treatment.
The GLP-1r signaling system has been the centerpiece of diabetes drug development in the past 
decade with two classes of drugs, peptide agonists with extended activity in circulation and DPP-4 
inhibitors that prolong the action of endogenous GLP-1.  The effectiveness of these agents attests to the 
importance of GLP-1 signaling in glucose regulation.  The central hypothesis guiding this grant proposal is 
that fasting GLP-1 action, mediated by paracrine effects of α-cell GLP-1, contributes to the control of 
insulin secretion and the β-cell compensation for insulin resistance and glucose intolerance.  Testing this 
hypothesis is important for clarifying the mechanism of action of a vital pathway regulating β-cells and for 
refining the use of GLP-1 signaling for the treatment of diabetes and pre-diabetes.    
Design & Procedures
Describe the study, providing detail regarding the study intervention (drug, device, physical 
procedures, manipulation of the subject or the subject’s environment, etc.). Discuss justifications for 
placebo control, discontinuation or delay of standard therapies, and washout periods if applicable. 
Identify procedures, tests and interventions performed exclusively for research purposes or more 


frequently than standard of care. Include alternative therapies, concurrent therapies discontinued per 
protocol, risk benefit ratio, and use of tissue/specimens. Discuss monitoring during washout periods if 
applicable. Include brief description of follow-up, if any.
The entire study (Aims 1-3) will enroll up to 60 participants. While we anticipate that the screening blood 
draw will yield a number of screen failures, we expect that approximately 20-25 subjects will complete 
AIM-2 and 15-20 will complete AIM-3 of the study protocol. All study visits and procedures will take place 
in the Human Physiology Testing Core shared resource facilities located at the Duke Center for Living 
campus.
AIM-2
Approximately 20-25 healthy subjects will complete Aim-2 of the study and these participants will have 
three study visits – a baseline consent visit, and two infusion procedures. 
Visit 1:  The consent visit will take place at the Duke Center for Living and will last approximately two 
hours. If the participant has given prior verbal consent during the phone screening process, they will fast 
(nothing but water) for 12 hours before this visit and their body mass index (composed of weight and 
height measures) will be checked to see if it falls within the inclusion range.  Daily medications may be 
taken.   We will explain the study and the consent form in detail.  Upon signing the consent form, a sample 
of blood (one teaspoon) will be drawn to measure hemoglobin A1c (HbA1c), fasting glucose level, kidney 
function (i.e. eGFR) and blood count (i.e. hematocrit) are within normal limits to ensure that the 
participant meets inclusion criteria.  If these values are within the range listed below for the appropriate 
cohort, the participant will be scheduled for Visit 2. 
If a woman is of childbearing potential, a blood (using one teaspoon of blood drawn by a needle-stick) 
pregnancy test will be conducted and it must be negative prior to continuation in this study.   
Visits 2 and 3: Each participant will have two 4-hour hyperglycemic clamp procedures with the addition of 
the GLP-1 receptor antagonist Exendin-9 (Ex-9) to determine the 
. During both visits, 
fasting GLP-1 effect
the participants will receive an infusion of Ex-9 beginning at the 60-minute mark of the clamp procedure; 
this will allow a comparison of glucose-stimulated insulin secretion with and without GLP-1 action, a 
parameter we have termed the 
. Throughout the glucose infusions during visits 2 and 
fasting GLP-1 effect
3, 1 ml blood samples will be taken every 5-10 minutes beginning at the -15 minute time-point and 
continue until the end of the 120 minute procedure. These samples will be tested immediately with a YSI 
glucose analyzer and the values used to adjust the glucose infusion rate to maintain stable blood glucose 
concentrations (a glucose clamp) throughout the experiment.
Subjects will report to the Duke Center for Living after a 12-hour fast. Study staff will obtain the 
participant’s current body weight.  At Visit-2 a skin test to confirm they do not have any sensitivity to the 
Ex-9 investigational compound that will be used during both visits 2 and 3 will be administered. The skin 
test is specified by the FDA as a precaution for immediate hypersensitivity; 5 ng of Ex-9 is injected 
intradermally and subjects are monitored for 30 minutes for a wheal/flare reaction.  This skin test must be 
negative in order to continue with the study testing. Once a negative skin test has been recorded, the 
participant will not need to be retested for sensitivity to Ex-9 at visit 3.
Subjects will have two intravenous catheters placed, one in each arm: 1) for blood drawing, and 
2) for the infusion of test materials. The arm for blood draws will be heated to maintain blood flow and
arterialize venous blood. Four (4.0 ml) fasting blood samples will be taken at -15, -10, -5 and 0 minutes
and then a variable infusion of 20% dextrose will begin to clamp the blood glucose at basal +3 mM for 120
minutes. This hyperglycemic clamp will be stabilized over 60 minutes and 4.0 ml blood samples will be
taken every 5-10 minutes during this period to establish baseline insulin secretion and active GLP-1
levels.  During these visits, participants will receive an infusion of Exendin-9 (750 pmol/kg/min) between
the 60 and 120 minute time-points of the clamp procedure.  For consistency, blood draws (4.0 ml) will
occur every 5-10 minutes between the 60 and 120 minute time-points of both visit 2 and 3.  Upon
completion of the final 60-minute interval for the Ex-9 infusion, both the infusion and clamp procedures
will be complete. One additional blood sample (1 ml) will be drawn post the assessment to be tested
immediately with the YSI glucose analyzer to ensure glucose levels are within normal limits. 
 will
Visit 3
occur at least one week later and will be a repeat infusion procedure identical to 
  The total amount
Visit 2.
of blood collected during each of these visits will be a maximum of ~141 ml (~9.5 Tbsp).
    We will aim to complete studies in 10-15 subjects before and after approximately one week of 
dexamethasone(Dex) treatment to induce insulin resistance.  Insulin resistance in response to a short-
course (4 mg daily) of supraphysiologic glucocorticoids is an established research procedure and is 
completely reversible over the course of exposure we are proposing. However, the rate of reversal is 
variable among subjects preventing a straight counter-balanced allocation to treatment (i.e. half of 
subjects getting Dex before the first study and half before the second). Therefore we will enroll 6-10 
subjects as controls for effects of time and repeat testing. These individuals will have identical clamps with 
no dexamethasone at approximately 1 week intervals and will be determined at random by study staff.


AIM-3
Approximately 15-20 subjects will complete AIM-3 of the study and these participants will have two study 
visits – a baseline consent visit, and one infusion procedures. 
Visit 1: Visit 1 is a baseline study visit during which the following will take place: 
• Potential subjects will review and sign the consent form with study staff.
• The participant’s current medications will be recorded for review by study staff.
• A screening blood draw will be obtained to measure fasting glucose, HbA1c, hematocrit, and eGFR.
If a woman is of childbearing potential, a blood (using one teaspoon of blood drawn by a needle-stick) 
pregnancy test will be conducted and it must be negative prior to continuation in this study.
Visit 2: Similar to Aim 2, each participant will have a 4-hour hyperglycemic clamp procedure with the 
addition of the GLP-1 receptor antagonist Exendin-9 (Ex-9) to determine the 
. The 
fasting GLP-1 effect
participants will receive an infusion of Ex-9 beginning at the 60-minute mark of the clamp procedure. 
Throughout the glucose infusion during visit-2, 1 ml blood samples will be taken every 5-10 minutes 
beginning at the -15 minute time-point and continue until the end of the 120 minute procedure. These 
samples will be tested immediately with a YSI glucose analyzer and the values used to adjust the glucose 
infusion rate to maintain stable blood glucose concentrations (a glucose clamp) throughout the 
experiment. 
Subjects will report to the Duke Center for Living after a 12-hour fast. Study staff will obtain the 
participant’s current body weight.  At Visit-2 a skin test to confirm they do not have any sensitivity to the 
Ex-9 investigational compound that will be used during the infusion will be administered. The skin test is 
specified by the FDA as a precaution for immediate hypersensitivity; 5 ng of Ex-9 is injected intradermally 
and subjects are monitored for 30 minutes for a wheal/flare reaction.  This skin test must be negative in 
order to continue with the study testing. 
Subjects will have two intravenous catheters placed, one in each arm: 1) for blood drawing, and 2) for the 
infusion of test materials. The arm for blood draws will be heated to maintain blood flow and arterialize 
venous blood. Four (4.0 ml) fasting blood samples will be taken at -15, -10, -5 and 0 minutes and then a 
variable infusion of 20% dextrose will begin to clamp the blood glucose at basal +3 mM for 120 minutes. 
This hyperglycemic clamp will be stabilized over 60 minutes and 4.0 ml blood samples will be taken every 
5-10 minutes during this period to establish baseline insulin secretion and active GLP-1 levels.  During
these visits, participants will receive an infusion of Exendin-9 (750 pmol/kg/min) between the 60 and 120
minute time-points of the clamp procedure. For consistency, blood draws (4.0 ml) will occur every 5-10
minutes between the 60 and 120 minute time-points.  Upon completion of the final 60-minute interval for
the Ex-9 infusion, both the infusion and clamp procedures will be complete. One additional blood sample
(1 ml) will be drawn post the assessment to be tested immediately with the YSI glucose analyzer to ensure
glucose levels are within normal limits. The total amount of blood collected during this visit will be a
maximum of ~141 ml (~9.5 Tbsp).
Selection of Subjects
List inclusion/exclusion criteria and how subjects will be identified.
AIM-2 SELECTION OF HEALTHY SUBJECTS: Study recruitment will occur primarily through 
advertisements via Duke websites, as well as local newspapers (e.g. Independent, Durham Herald, Duke 
Chronicle); and social media venues (e.g. DMPI Facebook page, Instagram), will be used as well as use of 
our Duke Health and Exercise Research Trials registry. Study recruitment will also take place at public 
venues (e.g. Duke Farmer’s Market or Community Health Fairs). Potential subjects for this study will be 
directed to contact the study staff to obtain more information about the study, if interested. Study 
participants will also be recruited using flyers posted across the Durham VAMC hospital and Duke hospital
/clinic facilities, inviting interested individuals to contact the study recruitment coordinator for additional 
information.
Inclusion criteria:  
Healthy adults age 20-45 years
Male or female
Body Mass Index (BMI) ≤ 35.0 kg/m  
2
HbA1c ≤ 5.7%


Ability to speak and understand English
    
Exclusion criteria: 
Uncontrolled high blood pressure
Diabetes or use of diabetes medications such as insulin or metformin
Evidence of active heart disease or heart failure
Lung disease or COPD 
Malabsorptive GI disease, such as celiac disease, or gastric bypass
Significant hepatic disease
Kidney disease or renal insufficiency (eGFR < 60 mL/kg/min)
Untreated anemia (hematocrit < 34%) as measured at screening visit
Pregnant females
Active substance abuse
Chronic use of oral steroid medications such as prednisone and hydrocortisone
Apparent sensitivity to any of the study peptides as determined by the skin test
Diagnosis or h/o PTSD
Active mental health disorders such as depression, or as a result of Traumatic Brain Injury (TBI)
Exclusion criteria will primarily be identified by way of a phone screen process conducted by the coordinator, 
however medical record review may be used to confirm the absence of a diabetes mellitus diagnosis or other 
medical conditions listed as exclusion criteria.
AIM-3 SELECTION OF SUBJECTS:
 
Selection of Non-Diabetic Subjects: Study recruitment will occur primarily through advertisements via 
Duke websites, as well as local newspapers (e.g. Independent, Durham Herald, Duke Chronicle); and 
social media venues (e.g. DMPI Facebook page, Instagram) will be used as well as use of our Duke Health 
and Exercise Research Trials registry. Study recruitment will also take place at public venues (e.g. Duke 
Farmer’s Market or Community Health Fairs). Potential subjects for this study will be directed to contact 
the study staff to obtain more information about the study, if interested. 
Study participants will also be recruited using flyers posted across the Durham VAMC hospital and Duke 
hospital/clinic facilities, inviting interested individuals to contact the study recruitment coordinator for 
more information. In addition, the VA Informatics and Computing Infrastructure (VINCI) will be used to 
identify potential subjects that meet the inclusion criteria. The study staff will pre-screen subjects listed on 
the VINCI reports in CPRS and contact those subjects identified to meet the inclusion criteria. Recruitment 
may occur through the VA Endocrinology practice and primary care clinics.
Inclusion criteria - 
:
Non-Diabetic Subjects
Adults age 18-65 years
Male or female
Body Mass Index (BMI) 25-40 kg/m   
2
HbA1c ≤ 6.5% as measured at screening visit
No Diabetes or use of diabetes medications such as insulin or metformin
Ability to speak and understand English
Selection of T2DM Subjects: Methods for subject recruitment will be similar to the non-diabetic cohort 
with additional recruitment through the VA Endocrinology practice and primary care clinics, as well as 
potential assistance from search results from Slicer Dicer and our Duke Health and Exercise Research 
Trials or DCRU subject registries. 
Inclusion criteria - T2DM Subjects:
Adults age 18-65 years
Male or female
Body Mass Index (BMI) 25-40 kg/m2
HbA1c ≤ 7.5% plus a diagnosis of Type II diabetes managed by either Metformin, Sulfonylurea, or 
diet and exercise
Exclusion criteria:  ALL SUBJECTS
Uncontrolled high blood pressure
Evidence of active heart disease, unstable angina or heart failure
Lung disease or COPD
Malabsorption or GI disease, such as celiac disease, or gastric bypass
Significant hepatic disease
Kidney disease or renal insufficiency (eGFR < 60 mL/kg/min)


Untreated anemia (hematocrit < 34%) as measured at screening visit
Pregnant females
Active substance abuse
Chronic use of oral steroid medications such as prednisone and hydrocortisone
Apparent sensitivity to study peptides as determined by the skin test
Diagnosis or h/o PTSD
Active mental health disorders such as depression, or as a result of Traumatic Brain Injury (TBI)
Exclusion criteria will primarily be identified by way of a phone screen process conducted by the coordinator
/study staff, however medical record review for both cohorts may be used to confirm the absence of other 
medical conditions listed as exclusion criteria.
Subject Recruitment and Compensation
Describe recruitment procedures, including who will introduce the study to potential subjects. Describe 
how you will ensure that subject selection is equitable and all relevant demographic groups have access 
to study participation (per 45 CFR 46.111(a) (3)). Include information about approximately how many 
DUHS subjects will be recruited. If subjects are to be compensated, provide specific prorated amounts 
to be provided for expenses such as travel and/or lost wages, and/or for inducement to participate.
Study recruitment will occur primarily through advertisements via Duke websites, as well as local 
newspapers (e.g. Independent, Durham Herald, Duke Chronicle); and social media venues (e.g. DMPI 
Facebook page, Instagram), will be used. Study recruitment will also take place at public venues (e.g. 
Duke Farmer’s Market or Community Health Fairs). Potential subjects for this study will be directed to 
contact the study staff to obtain more information about the study, if interested. While the study staff will 
be a Duke employee on a WOC agreement with the Durham VAMC, all phone calls will be made using a VA 
cell phone and data obtained during the phone screen will immediately be entered into a database on a VA 
encrypted laptop. When contacted, the study staff will confirm the participant meets most of the inclusion
/exclusion criteria before scheduling him/her for a consent meeting.  Potential subjects will attend a group 
study information session that will be conducted by the study coordinator or another member of key 
personnel. 
We will allow approximately 30 minutes at the end of the information session for the subjects to 
read the consent, ask questions in a separate one-on-one setting and decide whether or not they are willing to 
As many as 60 subjects may be enrolled in this entire study (AIMS 1-3) as we 
participate in the study.  
seek to identify 45 individuals (for both AIM-2 and AIM-3 combined) that meet all inclusion/exclusion 
criteria and complete the study. Participants will receive $125 for the completion of each infusion 
procedure for a maximum of $250 (AIM-2) or $125 (AIM-3) upon study completion. Participant payments 
will be made through the Durham VA Medical Center’s cashier office. For Veterans with bank accounts, 
study staff members are required to complete a “Vendorizing Coversheet” with the participant, which 
includes bank account information. Any participant who does not have a bank account allowing direct 
deposit will be paid via checks issued through VA Financial Services in Austin, TX and mailed to their 
address on file. Participants will be informed that payment will be received approximately 4 to 6 weeks 
after it is requested.  For subjects who do not have a bank account or a physical address, payment will be 
made through the agent cashier.
There will be no costs to participants for any of the treatments or testing done as part of this research 
study. Immediate necessary care is available if an individual is injured because of participation in a 
research project. However, there is no provision for free medical care or for monetary compensation for 
such injury. During this study, hospitalizations or additional care beyond the scope of this study will be the 
responsibility of patients and/or their insurance company.
Subject’s Capacity to Give Legally Effective Consent


If subjects who do not have the capacity to give legally effective consent are included, describe how 
diminished capacity will be assessed. Will a periodic reassessment occur? If so, when? Will the subject 
be consented if the decisional capacity improves?
Subjects who have diminished capacity or who do not have the capacity to give legally effective consent 
will not be included. This assessment will be made by the study coordinator at the initial consent meeting.
Study Interventions
If not already presented in #4 above, describe study-related treatment or use of an investigational 
drug or biologic (with dosages), or device, or use of another form of intervention (i.e., either physical 
procedures or manipulation of the subject or the subject’s environment) for research purposes.
Risk/Benefit Assessment
Include a thorough description of how risks and discomforts will be minimized (per 45 CFR 46.111(a) (1 
and 2)). Consider physical, psychological, legal, economic and social risks as applicable. If vulnerable 
populations are to be included (such as children, pregnant women, prisoners or cognitively impaired 
adults), what special precautions will be used to minimize risks to these subjects? Also identify what 
available alternatives the person has if he/she chooses not to participate in the study. Describe the 
possible benefits to the subject. What is the importance of the knowledge expected to result from the 
research?
This study provides no potential benefit to the participant.  The following list describes potential risks 
associated with this protocol:
The potential risks from IV placement include momentary discomfort, bleeding, bruising, 
inflammation, and rarely infection or fainting.  A nurse will be present during the infusion 
procedures to monitor the IV access points and subjects will be asked to alert the study nurse if 
they experience any pain at the catheter site.
The risks from infusion of micromolar amounts of exendin-(9-39) are remote. In rare cases, nausea 
and vomiting have been reported as possible side effects for Ex-9.  The IND has been obtained 
from the FDA for the infusion of exendin-(9-39) [#65837] into humans. The Principal Investigator 
for the present study has used this peptide in several previous studies, in doses similar to those 
described in this application, with no attributable adverse effects observed.  Allergic reactions to 
synthetic peptides are possible; therefore skin testing will be performed on each subject to assess 
sensitivity prior to undergoing any clamp procedures. All peptides will be synthesized to GMP 
standards and documented sterile and pyrogen free.
The maximum amount of blood to be withdrawn during any one of these infusion procedures is 
approximately 141 ml and does not constitute an undue risk in persons with normal hematocrits.
At study entry, women of child-bearing potential will have a serum pregnancy test. The pregnancy 
test must be negative before the subject can undergo any study testing.
AIM-2: Some side effects of dexamethasone may include upset stomach, headache, dizziness, 
insomnia, restlessness, increased appetite or weight gain may occur. 
This is not a complete list of possible side effects. If the participant notices other effects not listed above, 
they will be advised to contact the study physician.
Costs to the Subject
Describe and justify any costs that the subject will incur as a result of participation; ordinarily, subjects 
should not be expected to pay for research without receiving direct benefit.
There will be no costs to participants for any of the treatments or testing done as part of this research 
study. Immediate necessary care is available if an individual is injured because of participation in a 
research project. However, there is no provision for free medical care or for monetary compensation for 
such injury. During this study, hospitalizations or additional care beyond the scope of this study will be the 
responsibility of patients and/or their insurance company.


Data Analysis & Statistical Considerations
Describe endpoints and power calculations. Provide a detailed description of how study data will be 
analyzed, including statistical methods used, and how ineligible subjects will be handled and which 
subjects will be included for analysis. Include planned sample size justification. Provide estimated time 
to target accrual and accrual rate. Describe interim analysis including plans to stop accrual during 
monitoring. Phase I studies, include dose escalation schema and criteria for dose escalation with 
definition of MTD and DLT.
AIM-2 – The primary outcome for visits 2-3 will be to measure the effects of Ex-9 on fasting glucose-
stimulated insulin secretion before and after experimentally induced insulin resistance.  The primary 
experimental variable for analysis will be C-peptide during the clamp. Mean values will be compared 
between the period of glucose only stimulation and glucose with Ex-9. For each subject in the active 
treatment arm data will be analyzed using 2-way ANOVA for repeated measures using time (0-60 vs 60-
120 min) and treatment (Dex vs no Dex) as the two factors. Based on our previous studies we expect a 
significant time effect due to Ex-9. If there is an interaction with treatment we would conclude that 
experimental insulin resistance influences the 
.  Data from control subjects will be 
fasting GLP-1 effect
analyzed identically; here we expect no interaction, indicating that the fasting GLP-1 is a stable 
measure.   In our previous study using Ex-9 during a glucose clamp, the average coefficient of variation in 
insulin concentrations at the conclusion of each step in the ramp was 30%.  Using this estimate of between 
subject variation, detecting a 20% difference between subsequent steps in the GLP-1 ramp with a power of 
80% and significance level of 0.05 will require 8 subjects. 
AIM-3 – The primary outcome measure for this study will be the fasting GLP-1 effect. This will be defined 
as the difference in steady state glucose-stimulated insulin secretion with and without Ex-9. The difference 
will be expressed as a percentage of glucose stimulated insulin secretion without Ex-9, and serve as the 
primary variable for comparison among the lean, obese and diabetes cohorts. 
Data & Safety Monitoring
Summarize safety concerns, and describe the methods to monitor research subjects and their data to 
ensure their safety, including who will monitor the data, and the frequency of such monitoring. If a 
data monitoring committee will be used, describe its operation, including stopping rules and frequency 
of review, and if it is independent of the sponsor (per 45 CFR 46.111(a) (6)).
As the PI has performed similar studies with humans in the past, a Data Safety and Monitoring Plan 
(DSMP) has already been established for studies involving synthetic peptides such as exendin-(9-39). This 
DSMP will continue for the oversight of this project.
No toxicities of exendin-(9-39) or GLP-1 have been described in previous studies in humans. For the 
purposes of this study, a serious adverse event is defined as any of the following: death, life threatening 
event, or an event resulting in permanent disability, hospitalization, or prolongation of a hospital stay. 
Thus, the intervention and protocol represents slightly more than minimal risk to the research subjects. 
Due to the low risk status of this study, the data safety monitoring plan (DSMP) for this project represents 
the monitoring for adverse events by the principal investigator (PI) in conjunction with a Safety Officer.
The Safety Officer for this trial will be William E. Kraus, MD. Dr. Kraus is a practicing Cardiologist who has 
experience in trial design and clinical trials involving diabetes and carbohydrate metabolism. He is familiar 
with the procedures used in this project and is very familiar with the mechanisms for reporting adverse 
events. As Safety Officer, Dr. Kraus will review the reports sent to him by the PI or his designees and will 
determine whether corrective action should be communicated to the PI, the IRB, the GCRC, NIH, and the 
FDA. The Safety Officer will be available to discuss glycemic control strategies with respect to the 
individual subject’s medical care goals and the goals necessary for the study. If the goals and strategies 
necessary for the study exceed those necessary for medical care or practical for that individual, the PI and
/or Safety Officer may choose to exclude the subject or discharge them from the study. Annually or at the 
completion of an Aim, the Safety Officer will complete a checklist that is used as a reminder regarding the 
functions he is to perform. We will prospectively collect data regarding potential adverse events during the 
course of the study and collate them on an annual basis. Since the PI and study team at the Center for 
Living will have frequent contact with the subjects, they are ideally suited to document the presence of 
Adverse Events.
There are a number of expected adverse events which will not be documented. These include mild 
hypoglycemic reactions, transient nausea and/or dizziness, and bruising/phlebitis at IV sites as these do 


not qualify as serious adverse events in this project. For the purposes of this study, a serious adverse 
event is defined as any of the following: death, life threatening event, or an event resulting in permanent 
disability, hospitalization, or prolongation of a hospital stay.  These will be documented and reviewed by 
the safety officer as they arise.
In this low risk protocol, it is very unlikely that excess adverse events would occur that would require 
stopping the study. As outlined elsewhere, adverse events will be monitored and reviewed by the Safety 
Officer who would make any determination regarding stopping the protocol because of unacceptable risks 
to subjects. In addition, it is very unlikely that new information would become available during the study 
that would affect the protocol's integrity. This information would be captured on the Safety Officer 
Checklist which is reviewed annually or at the completion of an Aim.
In accordance with federal regulations the PI will monitor for, review, and promptly report to the IRB, 
appropriate institutional officials, sponsor, coordinating center and the appropriate regulatory agency head 
all unanticipated problems involving risks to subjects or others that occur in the course of a subject’s 
participation in a research study, and all reportable AEs will be submitted per the DUHS IRB policies.
Describe Role of External Personnel:
Not applicable.