A Novel Cross-sectional Analysis of Insulin Sensitivity Among Adolescents and Young Adults With Type 1 Diabetes, MODY2, and Normal Controls: the Contribution of Hyperinsulinemia vs. Hyperglycemia to Insulin Resistance
Phase: PHASE1
Conditions: Type 1 Diabetes Mellitus, Maturity-Onset Diabetes of the Young, Type 2, MODY2, Insulin Resistance
Sponsor: Vanderbilt University

A NOVEL CROSS-SECTIONAL ANALYSIS OF INSULIN SENSITIVITY 
AMONG ADOLESCENTS AND YOUNG ADULTS WITH TYPE 1 
DIABETES, MODY2, AND NORMAL CONTROLS: THE CONTRIBUTION 
OF HYPERINSULINEMIA VS. HYPERGLYCEMIA TO INSULIN 
RESISTANCE
 
NCT02971202
          October 24, 2017

Protocol Version #:  10 
 
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Protocol Date: October 24, 2017 
 
 
 
 
 
 
 
A NOVEL CROSS-SECTIONAL ANALYSIS OF 
INSULIN SENSITIVITY AMONG ADOLESCENTS 
AND YOUNG ADULTS WITH TYPE 1 DIABETES, 
MODY2, AND NORMAL CONTROLS: THE 
CONTRIBUTION OF HYPERINSULINEMIA VS. 
HYPERGLYCEMIA TO INSULIN RESISTANCE 
 
Justin M. Gregory, M.D. 
Ian M. Burr Division of Endocrinology and Diabetes 
Department of Pediatrics 
Vanderbilt University Medical Center 
 
 
 
 
 
 
 
 
 
Alan D. Cherrington, Ph.D. 
 Professor, Molecular Physiology and Biophysics 
Jacquelyn A. Turner and Dr. Dorothy J. Turner Chair in Diabetes Research, Medicine 
Vanderbilt University 
 
Naji N. Abumrad, M.D. 
John L. Sawyers Professor of Surgery 
Department of Surgery 
Vanderbilt University Medical Center 
 
 
 

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Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Statistical Considerations 
11.0 Privacy/Confidentiality Issues 
12.0 Follow-up and Record Retention 
13.0 References 
 
Appendices 
Appendix A Pre-Study Glucose Log 
Appendix B Post-Study Glucose Log 

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1.0 
Background 
 
Type 1 diabetes mellitus (T1DM) is a chronic condition in which autoimmune pancreatic 
islet destruction leads to deficient insulin secretion resulting in hyperglycemia. Current 
therapy largely focuses on using intensive insulin regimens to reduce hyperglycemia to 
prevent micro- and macrovascular disease while minimizing hypoglycemia. Although 
these comorbidities are well-recognized,(1-7) insulin resistance (IR) is a consistent(8-12) 
and under-appreciated finding in T1DM, even among patients who lack traditional IR risk 
factors. In studies utilizing the insulin clamp to assess IR, insulin sensitivity was 
consistently lower in T1DM patients compared to their age, gender, and BMI matched 
controls without diabetes by 35-57%.(8-13) Because IR has been strongly and 
independently correlated with macrovascular disease in T1DM,(14-16) an increased 
understanding of its root cause is needed. While earlier investigations attributed T1DM 
IR to hyperglycemia, (17-22) more recent studies have shown little to no correlation 
between hyperglycemia and insulin sensitivity.(10, 11, 13, 23) Thus, the degree to 
which hyperglycemia contributes to IR in T1DM is unclear, and this uncertainty suggests 
that other factors contribute prominently. The proposed research will provide a 
framework for future therapeutic strategies to mitigate T1DM IR and associated 
macrovascular disease. 
 
2.0 
Rationale and Specific Aims 
 
The following research plan will determine whether IR in T1DM is predominantly an 
effect of chronic hyperglycemia, as is commonly accepted,(17-22) or a consequence of 
iatrogenic hyperinsulinemia in the peripheral circulation, as I alternatively hypothesize.  
Despite its independent contribution to micro- and macrovascular disease,(14, 16, 24) 
the underlying cause of IR has not been established nor have strategies to mitigate it 
been developed.  
 
Insulin therapy in T1DM attempts to achieve euglycemia but does so in an 
“unphysiologic” way, by delivering insulin into the subcutaneous tissue as compared to 
physiologic delivery directly into the hepatic portal circulation. Thus, peripheral insulin 
delivery must balance competing factors: 1) a sufficient amount of insulin must reach 
the liver to prevent unrestrained hepatic glucose production and resultant hyperglycemia 
and 2) simultaneously, an excess of insulin in the peripheral circulation must be avoided 
to prevent hypoglycemia, as I recently reported.(25) Although life-saving, peripheral 
insulin delivery in T1DM results in a loss of the normal insulin distribution; the 
physiologic state maintains insulin at 3-fold higher concentrations in the portal 
circulation compared with the peripheral circulation.(26-29) IR in T1DM could therefore 
occur in response to peripheral hyperinsulinemia, a mechanism that would protect 
against hypoglycemia and ensure adequate glucose delivery to the CNS.   
 
This protocol is designed to test these hypotheses:   
1) IR in T1DM is a homeostatic response to increased peripheral insulin concentrations 
resulting from peripheral insulin delivery and not significantly attributable to 
hyperglycemia, and  
2) IR in T1DM results primarily from peripheral tissue IR (especially muscle) and not 
primarily from hepatic IR.  

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To test this hypothesis, I plan to utilize the hyperinsulinemic,  euglycemic clamp (insulin 
clamp) to assess IR in a cross-sectional study of 3 groups of subjects:  
 
1) non-diabetic control subjects,  
2) patients with well controlled T1DM, and  
3) patients with glucokinase (GCK) mutations causing mature-onset diabetes of the 
young, type 2 (MODY2), a population that has hyperglycemia without 
hyperinsulinemia.  
 
As summarized in Table 1 and discussed in the following proposal, key metabolic 
differences between these 3 groups will enable distinction to be made between the 
relative contributions of peripheral hyperinsulinemia vs. hyperglycemia to IR in T1DM.   
 
Specific Aim 1: Determine whether whole-body IR in T1DM is secondary to 
peripheral insulin delivery (as opposed to hyperglycemia). This study is 
designed to select T1DM subjects with matched glycemic control to that of MODY2 
subjects. Importantly, because MODY2 patients retain pancreatic insulin secretion, the 
key factor influencing a difference in insulin sensitivity between these two groups will be 
that T1DM patients have iatrogenic peripheral hyperinsulinemia and portal 
hypoinsulinemia, while the insulin distribution remains normal in MODY2 (Table 1). 
Additionally, because there is no difference in insulin distribution between MODY2 and 
control subjects, the key factor influencing a difference in insulin sensitivity between 
these two groups is the presence of hyperglycemia in MODY2 and euglycemia in control.   
 
Specific Aim 2: Establish the tissue-specific distribution of IR in T1DM 
(muscle, fat, and liver). The tissue-specific site of IR in T1DM has not been well 
established. By utilizing a 2-step clamp procedure, I will be able to simultaneously 
assess whether T1DM IR is predominantly an effect of muscle IR (as I hypothesize) or 
whether hepatic IR also contributes to the whole body IR seen in this condition.  
 
A variation of the hyperinsulinemic, euglycemic clamp, the “pancreatic clamp technique” 
will be employed for the first time in T1DM and MODY2 subjects.  The technique 
employs a short-term somatostatin infusion to suppress endogenous insulin and 
glucagon release while these hormones are simultaneously replaced at a prescribed 
infusion rate.  Importantly, this approach ensures that insulin and glucagon, the key 
hormones influencing glucose metabolism, are equal between cohorts.  A failure to 
match these hormones would confound the study results, particularly the assessment of 
hepatic insulin resistance.  Somatostatin is a naturally occurring hormone, commercially 
Table 1: Key Between-Group Differences between control, MODY2, and T1DM subjects. Pe=peripheral, 
Po=portal. Shading is used to highlight similarities and differences between groups. Note: 1) MODY2 and T1DM 
subjects will be selected to have similarly mild hyperglycemia 2) Insulin distribution between peripheral and portal 
circulations will be normal in control and MODY2 subjects, but altered in T1DM. 

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available, and has been used in metabolism studies in the U.S. (30-36) and Europe (37-
41), but an FDA approval for clinical use in humans has never been pursued (largely 
because of the more favorable pharmacokinetic profile of its analog, octreotide).  An 
Investigational New Drug (IND) application to use somatostatin in this study is presently 
being reviewed by the FDA (IND #
). 
  
The proposed research will lay the groundwork for future testing of novel therapeutic 
strategies to restore the normal portal to peripheral insulin distribution can normalize 
insulin sensitivity in T1DM (e.g. hepatopreferential insulin analogs, intraperitoneal insulin 
delivery).  Further, it will clarify the pathophysiologic processes underlying IR in T1DM at 
a tissue-specific level.  
 
3.0 
Animal Studies and Previous Human Studies 
 
Many have thought that hyperglycemia per se was the predominant contributor to IR in 
T1DM.  Yki-Järvinen , Koivisto, et al., showed that short bouts of hyperglycemia (24 
hours) significantly reduced glucose uptake by ≈20% during clamp studies of T1DM 
patients compared to euglycemia of equal duration (18, 19).  The same group reported 
glucose disposal during hyperinsulinemic, clamp studies was inversely related to HbA1c 
in a cross sectional study of T1DM 53 patients with disease durations ranging from 2-32 
years (17).  The Pittsburg Epidemiology of Diabetes Complications Study found that 
HbA1c was one of the best predictors of insulin resistance in T1DM (42).   
 
The notion that hyperglycemia is the primary cause of IR in T1DM has been questioned 
in recent years, however.  No correlation was found between HbA1c or continuous 
glucose monitoring (CGM) data and peripheral glucose uptake in hyperinsulinemic, 
euglycemic clamps in the Coronary Artery Calcification in Type 1 Diabetes study (13).  In 
a later study, linear regression analysis showed that hyperglycemia accounted for only 
6.3% of the decrease in glucose uptake in their T1DM subjects compared to matched 
controls (10).  In a study of adolescents with T1DM, (11) no relationship between IR 
and HbA1c was found and in another (23) the correlation was weak.  Thus, the degree 
to which hyperglycemia contributes to IR in T1DM is unclear and this uncertainty 
suggests that other factors likely contribute prominently.   
 
IR in T1DM: A Homeostatic Response to Peripheral Hyperinsulinemia 
IR in T1DM can be alternatively hypothesized to be a homeostatic response to increased 
insulin concentrations.  Current therapy relies on subcutaneous insulin injection into the 
peripheral circulation to restore euglycemia in T1DM.  Although life-saving, a reversal of 
the normal insulin distribution occurs when insulin is delivered into the peripheral 
circulation, rather than more physiologically into the hepatic portal circulation with 
higher insulin concentrations in the peripheral circulation and lower insulin levels in the 
hepatic portal blood (26-28).  IR in T1DM could therefore be a compensatory 
downregulation of the glucose transport system to protect against hypoglycemia.  Two 
key studies support this explanation for T1DM IR: 
- 
Del Prato, DeFronzo, et al. examined the effect of sustained, physiologic 
euglycemic hyperinsulinemia on IR in 8 healthy subjects (43).  After performing 
baseline hyperinsulinemic, euglycemic clamps, subjects underwent 72-96 hours 
of continuous IV insulin infusion at 0.25 mU/kg/min with variable glucose 

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infusion to maintain fasting plasma glucose at 86 ± 2 mg/dL.  This led to a 3.3-
fold increase in the fasting insulin concentration.  After the 72-96 hour period, 
the hyperinsulinemic, euglycemic clamps were repeated.  During the 3-steps of 
the clamps, insulin sensitivity decreased by 25% (P<0.01), 40% (p<0.001), and 
20% (p<0.02) compared with the pre-treatment clamps.  These studies show 
that a relatively short period of physiologic hyperinsulinemia with sustained 
euglycemia can lead to a significant increase in IR. 
- 
Carpentier et al. studied insulin action in 16 T1DM patients who were recipients 
of combined kidney-pancreas transplantation with anastomosis of the pancreatic 
vein into either the systemic (KPT-S) or portal (KPT-P) circulation (44).  All study 
patients had normal fasting blood glucose (BG) levels and HbA1c and were 
matched for age, BMI, T1DM duration, immunosuppressant doses, and GFR.   
Using a stepwise, hyperglycemic clamp, KPT-S subjects had a ≈40% higher total 
area under the insulin secretion compared to the KPT-P and non-diabetic control 
groups.  Notably, insulin area under the curve was practically the same for KPT-P 
and control.  A second group of experiments utilized a 2-step hyperinsulinemic, 
euglycemic clamp.  Insulin sensitivity was 26% (p=0.07) and 32% (p=0.008)  
higher, respectively, in the KPT-P group than the KPT-S group. These data 
suggest that when baseline glycemic control and pulsatile insulin secretion are 
essentially equal (both T1DM groups had pancreas transplants), chronic insulin 
secretion into the portal circulation led to near-normalization of insulin sensitivity 
that was significantly better than chronic secretion into the peripheral circulation.   
Taken together, these two studies show 
that 1) peripheral hyperinsulinemia within 
the physiologic range induces whole-body 
IR independent of glycemia and 2) a 
restoration of pancreatic insulin secretion 
into the portal circulation normalizes IR in 
T1DM. 
 
Because of the complexity of insulin 
clamp studies in humans, I have learned 
this methodology in collaboration with Dr. 
Jason Winnick, a research assistant 
professor at Vanderbilt. I have performed 
nearly 50 insulin clamp studies to 
evaluating an exercise intervention 
program’s effect on hepatic IR in T2DM 
subjects. My experience in this study (as 
shown in Figure 1) illustrates my ability 
to detect changes in insulin sensitivity in diabetic patients with accuracy and precision 
using this approach. The experience gained from performing these studies makes me 
confident in my ability to perform the following proposed studies, under the continued 
guidance of my co-mentors, Drs. Naji Abumrad and Alan Cherrington.   
 
4.0 
Inclusion/Exclusion Criteria 
 
Figure 1: Hepatic insulin sensitivity before and 
after an exercise intervention program, quantified 
as the percent suppression of glucose production 
for a fixed insulin infusion rate. This data is 
intended to illustrate my ability to conduct the 
proposed experiments. CON=control subjects, 
EX=exercise subjects, PRE= pre-exercise 
intervention, POST= post-exercise intervention. 

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Inclusion and exclusion criteria for study participants are listed in Table 2 and will be 
determined at the initial screening visit. Subjects will be recruited from the Vanderbilt 
Eskind Diabetes Clinic, a large academic center that sees 4,000 patients with T1DM 
annually. T1DM subjects will be recruited to match MODY2 subjects within a HbA1c ± 
0.3%, age ± 5 years, and BMI ± 1.5 kg/m2. Control subjects will match MODY2 subjects 
within a BMI of ± 1.5 kg/m2 and age ± 5 years.  
 
Table 2: Inclusion and Exclusion Criteria for Study Participants 
 
 
5.0 
Enrollment/Randomization 
 
To test the aims of this proposal, the hyperinsulinemic, euglycemic clamp will be used to 
assess IR in three groups of  subjects:  
 
1) non-diabetic control subjects, 

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2) patients with well-controlled T1DM, and 
3) patients with GCK mutations causing MODY2. 
 
This is a novel paradigm using a cross-sectional study design comparing these three 
groups. 
 
After initial contact with interested individuals suggests they might be candidates for 
enrollment in the study, they will be invited for an initial screening visit in the Vanderbilt 
Clinical Research Center (CRC).  A history and physical exam will be performed and 
baseline clinical factors will be assessed.  If the potential subject meets 
exclusion/inclusion criteria for enrollment, they will be invited to enroll in the 
hyperinsulinemic, euglycemic clamp study as outlined in section 6.0.  The clamp study 
will occur within one month of the screening study. In the event a participant 
successfully meets exclusion/inclusion criteria for study and the clamp study is started 
but a technical issue precludes completion of the clamp study (e.g. unable to maintain 
IV access), an abbreviated screening will be pursued as outlined in section 6.2.2.5.  
 
A detailed description of special protection for adolescents as research subjects follows 
in section 7.1 below. 
 
The Vanderbilt CRC is located 
 
Nashville, TN 
 
 
6.0 
Study Procedures 
 
6.1 
Recruitment 
Multiple tools available to VUMC investigators will be utilized to identify 
prospective study participants.  These include Research match, Subject 
locator, social media platforms, and My Research at Vanderbilt.  Flyers 
will be posted and an institution-wide mass emailing will be used. 
Intramural and extramural endocrinology colleagues at Vanderbilt and in 
the region will be informed of this study and asked to refer potential 
subjects. The Vanderbilt Eskind Diabetes Clinic is a large academic center 
that sees approximately 4,000 patients with T1DM annually. Also, we will 
post our research flyer on social media platforms (e.g. Facebook and 
Twitter) to target further potential T1DM participants. For example, the 
JDRF Facebook pages have a large number of followers across the state 
of Tennessee who might be interested in participating in our research.  
 
If unable to recruit an adequate number of MODY2 subjects from 
Vanderbilt and other endocrinology clinics in Tennessee with which we 
have relationships established, a collaborative relationship will be pursued 
with colleagues at the University of Chicago, the leading institution in the 
study of monogenic diabetes in the U.S.  Additionally, we will post our 
research flyer on social media platforms (e.g. Facebook and Twitter) to 
target potential MODY2 participants. For example, the Monogenic 

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Diabetes Awareness Facebook page is followed by over 1,000 individuals 
who might be interested in participating in our research. 
 
Once the research team becomes aware of a potentially interested 
subject, a phone call will be made to make an initial assessment of 
whether the subject might be eligible to participate in the study.  Study 
personnel will ask the potential subject to share his or her: 
- diagnosis of T1DM, MODY2, or that the individual is interested in 
being a control subject 
- age 
- weight 
- height 
- most recent HbA1c (if applicable) 
- diabetes duration (if applicable),  
- medications 
- whether the potential subject has had severe hypoglycemia in the 
past 3 months 
- whether the potential subject has had any trips to the emergency 
department for poor glucose control in the past 6 months 
- if the patient has had GCK genetic sequencing confirming a 
diagnosis of MODY2 (if applicable) 
 
If this conversation reveals no reasons the potential subject should be 
excluded based on the criteria in table 2, he or she will be invited to 
participate in an initial screening visit. 
 
6.2 
Study visits: Each subject will participate in two study visits. 
 
6.2.1 Study visit 1: Initial screening 
All subjects will be asked to arrive at the CRC at 8 AM on the 
morning of study visit 1.  Participants will be asked to begin 
fasting except for water 8 hours before the visit.  T1DM subjects 
will be instructed beforehand that if they have hypoglycemia 
during the overnight fast to use the commonly known “Rule of 15” 
with glucose tablets 
(http://www.joslin.org/info/how_to_treat_a_low_blood_glucose.ht
ml) to correct their hypoglycemia.   
 
Upon arrival, the following will take place: 
 
- 
Consent/Assent: The PI or designated Key Study Personnel 
will obtain consent/assent from all participants. 
Consent/assent will be obtained in a private room in the CRC 
prior to beginning study procedures. The consent and assent 
form will be provided to the subject and family (if applicable) 
for review prior to the visit. The PI or designated Key Study 
Personnel will review the consent/assent forms with the family 

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in detail and provide time for questions to be answered. A 
copy of the consent/assent form will be provided. 
 
- 
History and Physical Exam:  Each subject’s clinical history 
will be reviewed by the PI (or a designated KSP who is an 
M.D., Nurse Practitioner, or Physician’s Assistant) and a 
physical exam will be performed.  Anthropometric 
measurements will be taken with the assistance of CRC staff 
or KSP.  If at this point the subject continues to meet inclusion 
and criteria (table 2), he or she will proceed with the 
remainder of visit 1. 
 
- Resting Metabolic Rate (RMR):  Resting metabolic rate 
(RMR) will be measured using indirect calorimetry (45). 
Subjects will recline in a hospital bed during this test. When 
the rate of oxygen consumption changes by less than 2% over 
a five minute period, it will indicate that the RMR has been 
achieved.  
 
- 
Blood and urine tests: each subject will have labs drawn 
following their 8-hour overnight fast.  These include a 
complete metabolic panel (screening for hepatorenal disease), 
hemoglobin (screening for anemia), c-peptide, insulin, HbA1c, 
lipid panel, and urine pregnancy test (for females). 
 
- DEXA:  Lean body and fat mass will be measured using dual-
energy X-ray absorptiometry (DXA) (Lunar Prodigy, enCore 
software version 10.5, GE Medical Systems).  
 
- 
Peripheral artery tonometry: Reactive hyperemia-
periopheral artery tonometry (RH-PAT) endothelial function 
testing will be used (Endo-PAT, Itamar Medical Ltd.) to assess 
risk of endothelial dysfunction and future premature 
cardiovascular disease. The subject will sit in a reclining chair 
with the hands at heart level and propped in a comfortable 
position such that the fingers are hanging freely. Fingertip 
probes are placed on both index fingers and pulse wave 
amplitudes are recorded for the duration of the study. After 5 
min of baseline measurement, arterial flow to the non-
dominant arm is occluded for 5 min using a BP cuff inflated to 
40 mmHg above systolic pressure. After the 5-min occlusion, 
the cuff is rapidly deflated to allow for reactive or flow-
mediated hyperemia. Pulse wave amplitudes are recorded for 
at least 5 min after the cuff is deflated. An integrated software 
program compares the ratio of arterial pressure in the two 
fingers before and after the occlusion to calculate the RH-PAT 
score.(46) 
 

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- VO2 max Testing:  Exercise testing will be performed to 
assess VO2 max.The Bruce protocol will be used, as described 
elsewhere (47). Participants will be asked to perform a 
progressive treadmill test for about 10 minutes (or until 
fatigued).  Attainment of a valid measure for VO2 max will be if 
two of the following three criteria are met: 1) a respiratory 
exchange ratio of 1.1 or greater, 2) attainment of a maximal 
measured heart rate within 10 beats/ min of the age predicted 
maximum (220-age), or 3) no change in oxygen consumption 
despite an increase in work load. T1DM participants will check 
their blood glucose prior to exercising.  If glucose is < 120 
mg/dL, a 15-30 gm carbohydrate snack will be provided. 
 
Following VO2 max testing, the subject will be provided with a 
meal from the CRC kitchen.  T1DM subjects will be instructed to 
resume their routine insulin regimen.  Patients will then be 
discharged home.  Once the data is collected from the screening 
visit and inclusion/exclusion criteria are confirmed as being met, 
the subject will be contacted. Study visit 2 will be scheduled 
within one month of study visit 1. 
 
Premenopausal females will be scheduled for their study during 
the follicular phase of their menstrual cycle (day 2-10) to reduce a 
potential confounder of insulin sensitivity.(48)  
 
T1DM and MODY2 subjects will be asked to perform an 8-point 
self-monitored BG profile (before meals, 2 h. post-prandial, 
bedtime, and 2 AM) for three days leading up to the clamp study. 
These participants will record this information on a glucose log 
designed for the study (see appendix). All participants will refrain 
from vigorous exercise and consume an isocaloric diet.  MODY2 
subjects from out of the region (if needed) may be required to 
consume the standard diet for less than 3 days to accommodate 
travel needs. 
 
6.2.2 Study visit 2: Hyperinsulinemic, euglycemic clamp 
 
Study subjects will undergo a two-step clamp as depicted in 
figure 2.  
 

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Figure 2: Hyperinsulinemic, euglycemic clamp protocol. Insulin infusion rates are predicted to cause a 3 and 10-fold rise in 
plasma insulin levels in control subjects.  In a 70 kg, 1.73 m2 individual, 12 mU/m2/min would approximately equal 0.3 mU/kg/min 
and 40 mU/m2/min would approximately equal 1.0 mU/kg/min.

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6.2.2.1 
Overnight CRC stay 
 
Subjects will be admitted to the Vanderbilt CRC on the 
night before the study and begin fasting except for 
water at 10 PM. All female subjects will have a second 
hCG measured to confirm the absence of pregnancy 
upon arrival to the CRC.   
 
T1DM and MODY2 subjects will then be given a 
continuous IV infusion of regular human insulin 
(recombinant DNA origin), titrated by the protocol of 
White et al. (49) as shown in Table 3.  
 
Table 3: Algorithm for Maintenance of Overnight 
Euglycemia 
Glucose reading 
(mg/dL) 
Insulin Infusion rate 
(U/h) 
above 250 
5 
201-250 
4 
171-200 
3 
141-170 
2 
121-140 
1.5 
101-120 
1 
81-100 
0.8 
61-80 
0.5 
under 60 
0 
 
Glucose will be checked on an hourly basis using a 
calibrated, hospital bedside glucose monitor.  Testing 
this protocol in 25 subjects with T1DM, White et al. 
reached and maintained near-euglycemia (101 ± 2 
mg/dL) in the fasted state. Glucose fell to below 60 
mg/dL on six occasions and to below 50 mg/dL on only 
one occasion. T1DM and MODY2 study patients should 
be at euglycemia when the clamp study begins 
(matching them with non-diabetic controls) and the 
overnight-titrated fasting insulin infusion rate should 
approximate the peripheral insulin infusion rate needed 
for euglycemia in each patient. Should glucose fall to < 
75 mg/dL the PI may elect to infuse a small dose of 
20% dextrose to prevent hypoglycemia.  Should 
glucose fall to < 55 mg/dL and the patient experience 
symptoms of hypoglycemia, 20% dextrose will be at 
bedside to treat the hypoglycemia (e.g. 75 mL of 20% 
dextrose delivers 15 gm of glucose).  If glucose were 
to fall to < 55 mg/dL, the following morning’s clamp 
study would need to be postponed to a later date 

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Protocol Date: October 24, 2017 
because of the confounding influence of the counter-
regulatory response to hypoglycemia.   
 
The PI will be physically present in the CRC for at least 
the first 3 overnight insulin infusions (T1DM or MODY2 
subjects) to monitor and adjust the insulin infusion 
from the protocol if needed.  Thereafter, the PI and 
CRC nurse manager will confer on the need for the PI 
(or a qualified M.D., N.P., or P.A.) to be physically 
present for the overnight insulin infusions based on the 
safety profile of the preceding IV insulin infusions. If 
the PI is not physically present, he will be readily 
available via telephone pager and will be within 20 
minutes of the CRC.   
 
Control subjects will be admitted on the night prior to 
the study, begin fasting at 10 PM, but no insulin will be 
infused until the start of period 1.   
 
6.2.2.2 
Hyperinsulinemic, euglycemic clamp  
 
As depicted in figure 2, each experiment will consist of: 
1. a 90 min equilibration period for [6,6-2H]-
glucose,  
2. a 30 min basal sampling period (control),  
3. a 30 min period to obtain muscle biopsy (Bx) 
#1; then  
4. 2 consecutive, 150 min experimental periods 
where increasing IV infusions of regular human 
insulin are given, and  
5. a 30 min period for Bx #2.  
 
Somatostatin will be infused into a peripheral vein to 
allow for a pancreatic clamp. Simultaneously glucagon 
will be infused into a peripheral vein at a rate chosen 
to approximate basal concentrations of glucagon at the 
liver, preventing a fall in glucagon that would 
otherwise occur secondary to the hyperinsulinemia and 
somatostatin. As noted previously, this allows for 
insulin and glucagon concentrations to be identical 
between test subjects, enabling a direct assessment of 
hepatic insulin sensitivity (50).  In the United States, 
numerous groups currently use somatostatin for the 
purpose of “clamping” pancreatic hormone secretion 
(30-36) and it is used extensively in Europe for the 
same purpose (37-41).  
 

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Protocol Date: October 24, 2017 
Plasma glucose will be monitored every 10-30 minutes 
during the equilibration, control, and Bx1 periods, then 
every 5-10 minutes during the 2 consecutive 150 min 
experimental periods and Bx2.  The YSI Glucose 
Analyzer (Yellow Springs, OH), the gold standard 
instrument for glucose concentration analysis, will be 
used during the clamp study.  IV glucose will be 
infused to maintain plasma glucose at approximately 
90-100 mg/dL throughout the study.  At nine points in 
the study (3 times each during the control, sample 1, 
and sample 2 periods as in figure 2), a larger blood 
draw will be made from a second intravenous catheter 
to determine plasma concentrations of the following:  
- 6,6 2H-glucose 
- Insulin 
- Catecholamines 
- Glucagon 
- Cortisol 
- C-peptide 
- Free Fatty Acids 
- Lactate, glycerol, alanine (and possibly beta-
hydroxybutyrate) 
- Triglycerides 
These blood draws are calculated to equal just under 
160 mL in total blood volume. This includes the volume 
of blood that is wasted to clear the IV line with each 
blood draw. 
 
6.2.2.3 
Muscle biopsy 
 
Subjects will be asked to allow the study team to 
perform two muscle biopsies during the study. If the 
subjects decline the muscle biopsy, they will still be 
allowed to participate in the hyperinsulinemic, 
euglycemic clamp. 
 
The muscle biopsies, Bx #1 and #2, will be obtained 
immediately before study period 1 and immediately 
following study period 2, respectively (figure 2). 
Percutaneous biopsies will be performed on the lateral 
portion of the quadriceps femoris muscle (vastus 
lateralis) using sterile technique. A minimum distance 
of 2 cm proximal or distal between the two sampling 
sites will be used to minimize specimen artifact caused 
by trauma, inflammation, and/or scarring. Local 
anesthesia with lidocaine 0.5-1% will be applied in two 
steps: first within the dermis and second into the 
muscle fascia before the biopsy is obtained. A 4-6 mm 

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Protocol Date: October 24, 2017 
incision through the skin will be made with a scalpel. 
Then we will insert a trocar into the incision site, 
pushing through the muscle fascia, and advance to 
ensure that the cutting chamber lies fully within the 
muscle. We will apply suction to withdraw the muscle 
tissue into the cutting chamber and then we will rotate 
the trocar clockwise 90 degrees to maximize the 
amount of muscle sample obtained. We will place 
sterile gauze and apply pressure with a cold compress 
for 10 minutes to minimize bleeding, inflammation, and 
pain. During and after the procedure, participants will 
be allowed to take acetaminophen on an as needed 
basis to minimize pain under the supervision of the PI. 
Muscle samples will be immediately blotted free of 
visible non-muscle tissue, flash frozen in liquid nitrogen 
and stored at -80°C until analysis. Samples will be 
analyzed using immunoprecipitation/immunoblot 
analysis. 
 
6.2.2.4 
Completion of clamp study 
 
Upon completion of the study, insulin will be 
discontinued and the study subject will be allowed to 
eat. Plasma glucose will be monitored every ten 
minutes for an additional hour and the glucose infusion 
rate will be steadily decreased to maintain glucose > 
120 mg/dL.  Patients taking insulin at home will be 
instructed to resume their home regimen, however 
they will be instructed to check glucose before dinner, 
bedtime, and at 2 AM following the study.  These 
glucose readings will be recorded on a 24-hour post-
study glucose log (see appendix). If glucose is < 120 
at any of these checks, they will consume an additional 
15-30 gm of glucose with 5-15 gm of protein to 
prevent hypoglycemia.  As an example one Luna bar 
has 26 gm of carbohydrates and 9 grams of protein.  
By the following morning, risk of hypoglycemia as a 
result of participating in the clamp study will be no 
greater than their baseline risk. 
 
After local anesthesia from the muscle biopsy wears 
off, the subject may experience soreness for less than 
a week; during this time the study team will 
recommend subjects use acetaminophen 650 mg every 
four hours as needed for pain or discomfort, with no 
more than 2,000 mg in a 24 hour period. The study 
team will instruct the participants and parents of 
adolescent participants on post-procedural wound 

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Protocol Date: October 24, 2017 
management.  We will recommend that subjects 
minimize walking on the day of discharge from the 
study; activity should gradually be resumed following 
the procedure. Vigorous activity or sport should be 
avoided for the first few days and resumed cautiously 
as discomfort subsides.  In addition to contacting the 
subject’s family on the day following the study to 
review blood glucose numbers, the study team will 
check on the status of wound healing during the call 
on the day following the study as well as 5-7 days after 
the study. The subject will be instructed to notify their 
primary care physician if they have marked swelling 
and discomfort of the whole muscle (beyond the 
anticipated bruising); if the biopsy site is painful more 
than a week after the biopsy was taken; if the biopsy 
site appears swollen, purulent, or fluctuant; if there is 
persistent oozing from the biopsy site; or if a 
temperature of >= 102 degrees F develops.   
 
6.2.2.5 
Rescheduling clamp study after a technical issue 
 
It is possible once a clamp visit has begun a technical 
issue will prevent the successful completion of the 
study. For example, IV access may be lost during the 
study or otherwise uneventful hypoglycemia (glucose < 
55 mg/dL) may occur for those participants receiving 
an overnight IV insulin infusion (section 6.2.2.1). In 
the event such a technical issue occurs, the PI may 
elect to reschedule a repeat clamp study with the 
following stipulations: 
 
A) The PI deems the technical issue poses no serious 
danger to the participant were it to occur again and 
the participant wishes to repeat the study 
B) The rescheduled clamp occurs within six months of 
the original screening visit 
C) An abbreviated screening procedure has been 
completed within one month of the repeated clamp 
visit. This abbreviated screening will re-assess 
inclusion/exclusion criteria (table 2) and safety to 
be restudied. The abbreviated screening will 
include: 
- 
Fasting blood tests: a complete metabolic panel 
(screening for hepatorenal disease), 
hemoglobin (screening for anemia), c-peptide, 
insulin, HbA1c, lipid panel 
- 
Urine pregnancy testing: on the night prior to 
the clamp in females 

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Protocol Date: October 24, 2017 
- 
History and Physical Exam: the participant’s interval clinical 
history will be reviewed by the PI (or a designated KSP who is 
an M.D., Nurse Practitioner, or Physician’s Assistant) on the 
evening prior to the clamp study and a physical exam will be 
performed.  Anthropometric measurements will be repeated 
with the assistance of CRC staff or KSP. 
7.0 
Risks 
 
During the participant consent/assent process study personnel will address potential 
discomfort and risks associated with the study protocol.  These will be included in the 
consent/assent form.   
 
The following risks associated with participation in this study are perceived as low:  
 
- venipuncture and intravenous angiocatheter placement: possible hematoma, site 
infection, nausea, and vasovagal syncope.  These risks will be minimized by 
performing the procedure with the subject seated and cleaning the site with the 
appropriate antiseptic prior to breaking the skin. 
 
- DEXA scan: studies of the radiation dose to patients from a total body DEXA scan 
have confirmed that patient radiation exposure is small compared to many other 
sources of exposure.  For the total body fast scan mode that will be employed 
the average skin entrance dose is 0.2 μSv (51, 52). By comparison a chest x-ray 
is associated with a patient dose of 50 μSv.   
 
The following risks associated with participation are additionally considered: 
 
7.1 
Exercise testing 
 
During the screening visit, subjects will perform an exercise test to 
measure VO2 max.  Because inclusion/exclusion criteria dictate that the 
subjects will be young (ages 13-35), not obese (BMI 20-28), and those 
patients with diabetes will have relatively ideal glycemic control (HbA1c 
6.0-8.0%), the risk of a cardiovascular complication of exercise is 
considered to be low.  The greatest risk of the exercise test is 
hypoglycemia in the T1DM patients who will have been fasting overnight.  
The patient will be asked to check their capillary glucose using their home 
meter immediately prior to exercise and if glucose is < 100 mg/dL, they 
will consume 15 gm of glucose immediately prior to the ~ 10 minute 
exercise test.  Immediately following this, all subjects will be provided 
with a meal, further minimizing exposure to hypoglycemia subsequent to 
the brief exercise test.   
 
7.2 
Hyperinsulinemia 
 
Subjects will be exposed to hyperinsulinemia during the clamp study, 
which could lead to hypoglycemia and hypokalemia. We will implement 
the following to minimize these risks: 

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Protocol Date: October 24, 2017 
 
- As shown in figure 2, the highest insulin rate used will be 40 mU/m2/min. 
This is the most frequently applied insulin infusion rate in human 
hyperinsulinemic, euglycemic clamp studies.(53) This infusion rate is 
anticipated to result in plasma insulin concentrations between 50 and 130 
μU/mL, (54) levels that are within the range of physiological 
hyperinsulinemia and comparable to concentrations seen after meal 
ingestion. (53, 55, 56) By comparison, the most commonly used insulin 
infusion rate in the treatment of diabetic ketoacidosis is 0.1 U/kg/hr.(57) 
In a 70 kg, 1.73 m2 individual, this infusion rate would equal 67 
mU/m2/min, over 50% higher than the 40 mU/m2/min rate used in this 
study. 
- Glucose will be monitored every hour during the overnight basal insulin 
infusion as outlined in 6.2.2.1.  Glucose will be monitored every 5-10 
minutes throughout the hyperinsulinemic clamp to guide the variable 
dextrose infusion and ensure euglycemia. Because glucose is monitored 
so frequently, a trend toward hypoglycemia would most likely be detected 
before hypoglycemia actually occurred. If hypoglycemia did occur, 
however, this frequent monitoring would allow for rapid detection of the 
low glucose concentration and rapid corrective treatment with an infusion 
of IV glucose (dextrose).  
- Concentrated dextrose will be at bedside to treat iatrogenic 
hypoglycemia. If hypoglycemia occurred it could rapidly be corrected.  
For example, a 75 mL IV bolus of 20% dextrose (15 gm of glucose) 
would be expected to bring glucose up above the hypoglycemic range in 
less than 5 minutes. This dextrose could be given into either of the two 
IV angiocatheters available during the study. 
- A licensed MD, NP, or PA will be on call throughout overnight CRC stay 
and bedside during clamp procedure 
- Potassium will be monitored using bedside istat monitor (and oral 
potassium replacement will be given as needed) 
- Glucose will continue to be monitored following the study for at least 1 
hour  
- Patients will be required to consume mixed meal immediately following 
clamp procedure. T1DM subjects will not be discharged unless blood 
glucose > 120. 
- Vital signs will be monitored throughout clamp procedure 
- Subjects will be counseled on the risk of late hypoglycemia the evening 
and night following the clamp. They will be advised of the importance of 
checking their blood glucose at bedtime and during the night to maintain 
a minimum of 120 mg/dL 
 
7.3 
Muscle biopsy 
 
Having a muscle biopsy may cause pain, soreness, bruising, or infection 
at the biopsy site.  Every effort will be made to prevent or minimize these 
side effects of the muscle biopsy:  

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Protocol Date: October 24, 2017 
- the biopsy will be of a large muscle that is easily identified on physical 
exam: the vastus lateralus of each lower extremity 
- sterile technique will be used to minimize risk of infection 
- local anesthesia with lidocaine (0.5-1.0%) will be used prior to the 
incision 
- after local anesthesia wears off, the subject may experience soreness for 
less than a week; during this time the study team will recommend 
subjects use acetaminophen 650 mg every four hours as needed for pain 
or discomfort, with no more than 2,000 mg in a 24 hour period. 
- the study team will recommend that subjects minimize walking on the 
day of discharge from the study; activity should gradually be resumed 
following the procedure. Vigorous activity or sport should be avoided for 
the first few days and resumed cautiously as discomfort subsides. 
- The subject will be instructed to notify their primary care physician if they 
have marked swelling and discomfort of the whole muscle (beyond the 
anticipated bruising); if the biopsy site is painful more than a week after 
the biopsy was taken; if the biopsy site appears swollen, purulent, or 
fluctuant; if there is persistent oozing from the biopsy site; or if a 
temperature of >= 102 degrees F develops.   
 
7.4 
Infusion of somatostatin  
 
The following studies will utilize an IV infusion of the peptide hormone 
somatostatin, a hormone not approved by the FDA for use in humans, 
but commonly and safely used in human glucose metabolism research in 
the United States (30-36) and Europe (37-41). A modification of the 
glucose clamp, commonly referred to as the “pancreatic clamp,” employs 
a short-term somatostatin infusion to suppress endogenous insulin and 
glucagon release while these hormones are simultaneously replaced at a 
prescribed infusion rate as shown in figure 2. Importantly, this approach 
ensures that insulin and glucagon, the key hormones influencing glucose 
metabolism, are equal between cohorts.  This key aspect of the 
pancreatic clamp is essential for these metabolic studies as it allows for 
equal plasma levels of both insulin and glucagon between T1DM patients 
who are not capable of insulin secretion and non-T1DM subjects who are. 
Failing to equalize levels of these hormones between test subjects would 
confound the results considerably. 
 
Other effects of somatostatin infusion include the inhibition of growth 
hormone secretion and the inhibition of gastric emptying. However, 
growth hormone is not known to be an acute modulator of glucose 
metabolism, thereby making this off-target effect of no meaningful 
consequence. Likewise, all subjects will be fasted while receiving the 
somatostatin infusion so its effect on gastric emptying will be of little 
consequence. We are not aware of somatostatin having been “withdrawn 
from the market” for any reason related to safety or effectiveness. 
Finally, somatostatin’s short half-life (1-3 minutes, (58) the primary factor 
driving the development and clinical application of longer-acting synthetic 

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Protocol Date: October 24, 2017 
analogs such as octreotide) ensures that these suppressive effects are of 
very short duration after the infusion is discontinued. 
Somatostatin used in these studies will be manufactured by Bachem 
Americas, Inc, in conformity with the requirements for Current Good 
Manufacturing Practice as specified in the International Conference on 
Harmonisation of Technical Requirements for Registration of 
Pharmaceuticals for Human Use.  Testing for determination of purity and 
uniformity was performed by HPLC.  The final drug product will be 
composed of 2.5 mg of somatostatin dissolved in 50 mL NS. The drug 
substance will be received by Vanderbilt’s Investigational Drug Service 
(IDS) as a powder and will be compounded to obtain the IV formulation. 
Vanderbilt’s IDS has extensive experience managing and compounding 
investigational agents for FDA-regulated studies and follows stringent 
procedures for aseptic compounding that adhere to USP<797> 
guidelines. The final IV formulation will be compounded by dissolving 
somatostatin in NS to achieve a 50 µg/mL solution. The IV formulation 
will be prepared sterile by utilizing a sterile, 0.2 micron filter, transferred 
into either sterile syringes or IV bags, and labeled as an investigational 
product. Filter integrity will be verified via bubble point testing as 
described in USP<797>.  Beyond use dating for the final product will be 
24 hours at controlled room temperature. A representative batch of 
somatostatin  4.5 mg in 50 mL NS was formulated as specified and 
demonstrated to be sterile.  A 1 mL sample from each drug product will 
be sent for endotoxin testing utilizing the Kinetic Turbidimetric Assay in 
compliance with USP<85>. An Investigational New Drug (IND) 
application for the use of somatostatin in this protocol is presently under 
review by the FDA (# 132209). 
 
7.5 
Infusion of glucagon 
 
Glucagon is a peptide hormone secreted by the alpha cells of the 
endocrine pancreas.  Its effect is generally opposite that of insulin as it 
raises hepatic glucose production by stimulating glycogenolysis, (59) a 
process that raises plasma glucose levels. It is FDA approved and 
commonly used in the treatment of hypoglycemia.  
 
As noted previously, glucagon will be infused into a peripheral vein at a 
rate chosen to approximate basal concentrations of glucagon at the liver, 
preventing a fall in glucagon that would otherwise occur secondary to the 
hyperinsulinemia and somatostatin. This allows for insulin and glucagon 
concentrations to be identical between test subjects, enabling a direct 
assessment of hepatic insulin sensitivity. 
 
Although rapid infusions of glucagon are commonly associated with 
nausea and headache, our infusion rate of 0.65 ng/kg/min has been 
shown to maintain glucagon at a consistent, basal concentration 
throughout pancreatic clamp studies. (33, 38, 60) Because glucagon will 

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Protocol Date: October 24, 2017 
remain at fasting levels throughout the study, these adverse effects are 
not anticipated.  
 
While glucagon potently raises plasma glucose in the hypoglycemic state, 
its glucose-raising affect is attenuated in the euglycemic and 
hyperglycemic state. (61) For this reason and because of IV glucagon’s 
short half-life (≈ 10 minutes), glucagon is not expected to cause 
iatrogenic hyperglycemia during or after the clamp study. 
 
 
7.6 
Special Protections for Adolescents as Research Subjects 
 
Because adolescent subjects between ages 13-18 are included in the 
T1DM and MODY2 cohorts, special consideration has been given to 
provisions designed to protect children as outlined in 45 CFR 46, Subpart 
D. 
 
7.6.1 Justification and rationale for inclusion of adolescent subjects with 
T1DM or MODY2 
 
T1DM 
 
Despite numerous advancements to reduce hyperglycemia in 
pediatric T1DM(62) since the publication of the groundbreaking 
Diabetes Control and Complications Trial in 1993,(63) 
macrovascular disease remains a profound contributor to excess 
mortality for the nearly 20,000 pediatric patients who are 
diagnosed with T1DM in the United States each year.(64)  Among 
present-day adolescents with T1DM, the estimated loss in life 
expectancy is 11 years for males and 13 years for females 
compared to their non-diabetic counterparts.(65) Overall, the 
largest percentage in estimated loss of life expectancy was related 
to ischemic heart disease (36% in men, 31% in women).(65)  
Even contemporary T1DM patients with ideal glycemic control 
(indicated by an average HbA1c ≤ 6.9%) remain at a 3-fold 
increased risk from cardiovascular disease death.(66) Thus, 
despite major advancements in the treatment of T1DM over the 
past 2 decades, the risk of death from macrovascular disease 
remains profoundly high. 
 
The atherosclerotic changes of macrovascular disease (e.g. 
increasing carotid intima media thickness) appear to begin in 
childhood and adolescence(67, 68) and morbidity and mortality 
risk is staggeringly high in younger adults.(69) As an example, 
among individuals ages 1-39, the standardized mortality risk (i.e. 
ratio between the observed number of deaths in a study 
population and the number of deaths that would be expected) for 
ischemic heart disease death is 8.9 (95% CI 6.2–12.9) in males 

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Protocol Date: October 24, 2017 
and an astounding 41.7 (95% CI 28.9–58.2) in females. Thus, it is 
clear that pathophysiologic mechanisms that contribute to 
macrovascular disease are operative early in the natural history of 
T1DM. These same early pathophysiologic mechanisms have not 
been identified, however.  
 
IR is a consistent(8-12) and under-recognized finding in T1DM, 
even among patients who lack traditional IR risk factors (such as 
those commonly seen in type 2 diabetes). Because IR has been 
strongly and independently correlated with macrovascular disease 
in T1DM,(14-16) an increased understanding of its root cause is 
needed. While earlier investigations attributed T1DM IR to 
hyperglycemia, (17-22) more recent studies have shown little to 
no correlation between hyperglycemia and insulin sensitivity.(10, 
11, 13, 23) Thus, the degree to which hyperglycemia contributes 
to IR in T1DM is unclear, and this uncertainty suggests that other 
factors contribute prominently. This study tests the hypothesis 
that iatrogenic hyperinsulinemia rather than hyperglycemia is the 
primary contributor to T1DM IR. To date only one study has 
examined pediatric T1DM patients with typical glycemic control, 
which found that adolescents were 37% less insulin sensitive than 
matched control subjects,(11) suggesting that exposure to IR 
begins early in the natural history of T1DM.  
 
To determine the root cause of T1DM IR and translate this 
information into therapies that would ameliorate IR in T1DM and 
its contribution to macrovascular disease, the mechanisms causing 
IR must be assessed early in the disease duration. This is 
because: 
 
1) T1DM IR appears to begin early in the disease process,(11, 
17)  
2) the incidence of T1DM onset is highest between ages 10-
14,(70) and  
3) at younger ages and shorter disease durations there are 
minimal confounding covariables that would contribute to IR 
at a molecular level. These confounding covariables include 
co-morbidities associated with T1DM such as diabetic 
nephropathy,(71) prolonged exposure to 
hypertriglyceridema,(72, 73) excessive BMI(74, 75) and 
visceral adiposity,(76, 77),  deleterious effects of aging and 
progressive inflammation on extracellular matrix 
remodeling,(78, 79) and increasing age per se.(80, 81)  
 
By studying young patients early in the disease duration, 
this investigation will not only more precisely determine 
whether iatrogenic hyperinsulinemia or hyperglycemia is 
the key modifiable risk factor to address in alleviating 

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Protocol Date: October 24, 2017 
T1DM IR, but will simultaneously define the mechanisms 
causing the IR at a molecular signaling level when 
confounding covariables are very minimal. This knowledge is 
critical towards guiding subsequent therapy to mitigate IR which 
will considerably reduce early mortality risk from macrovascular 
disease in the same subjects tested in the research.  
 
By studying younger patients early in the disease process, this 
investigation will minimize the confounding effect of these 
comorbidities and allow a more precise determination of whether 
iatrogenic hyperinsulinemia or hyperglycemia is the key modifiable 
risk factor to address in alleviating T1DM IR. With this knowledge, 
subsequent therapy can be directed towards mitigating IR and in 
so doing considerably reduce early mortality from macrovascular 
disease. Additionally, it will establish the presence or absence of 
other risk factors for macrovascular disease (e.g. dyslipidemia, 
endothelial dysfunction measured by peripheral artery tonometry, 
reduced exercise capacity) at an early point in disease duration. 
These data would inform subsequent longitudinal studies of 
macrovascular disease progression as a future direction. 
 
MODY2 
 
MODY2 is a less prevalent(82) form of diabetes seen in pediatrics, 
characterized by mild fasting hyperglycemia (110-145 mg/dL)(83) 
and mild chronic hyperglycemia (mean HbA1c  6.4-7.6%).(82, 84) 
An assessment of IR using the hyperinsulinemic, euglycemic 
clamp technique in this patient group has only been attempted 
once, was with technical limitations, and studied only one patient 
younger than age 18.(85) The proposed study will therefore 
considerably increase our knowledge of IR in MODY2 and 
represents the first-ever study of IR in this condition in the 
adolescent age group.  
 
As with adolescent T1DM subjects, this investigation of 
adolescents MODY2 will determine if IR is present when other, 
age-related confounding covariables that might influence IR are 
minimized. It will also provide valuable information about the 
presence or absence of other macrovascular disease risk factors at 
a relatively early point in the natural history of the condition. 
 
With these considerations in mind, this study proposes to test 
diabetic subjects in adolescence (ages 13-18, Tanner stage 5) 
and young adulthood (age 18-35).   
 
7.6.2 Assessment of risk to adolescent participants relative to 45 CFR 
part 46, subpart D 
 

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Protocol Date: October 24, 2017 
It is our impression that T1DM and MODY2 subjects would fall 
under 45 CFR 46.406, as outlined in Vanderbilt’s Human Research 
Protections Program Policy Number IX.A, section II.C.  As 
discussed in section 7.6.1, the proposed research is likely to yield 
generalizable knowledge about these adolescents’ condition. 
Specifically, 
 
1. The risk represents a minor increase over minimal risk. 
- The hyperinsulinemic, euglycemic clamp has been used 
safely and extensively in children and adolescents 
elsewhere.  The technique has been performed over 
one thousand times in healthy adolescent subjects at 
other institutions such as the University of 
Minnesota,(86-89) hundreds of times in overweight 
adolescent(90-92) and normal weight prepubertal 
subjects(93, 94) at the University of Pittsburgh, and a 
similar number of times in normal weight children and 
adolescents at the National Institutes of Health.(95, 
96) Our insulin maximal infusion rate of 40 mU/m2/min 
is approximately equal to the rate used in studies of 
healthy subjects at each of these institutions and half 
that used in overweight subjects studied at the 
University of Pittsburgh. 
- As outlined in section 7.2, extensive efforts have been 
employed to minimize risk of hypoglycemia and 
hypokalemia associated with hyperinsulinemia. 
- Although use of the “pancreatic clamp technique” 
employed here is not used in adolescent studies at 
these institutions, it is commonly in adult studies 
elsewhere with no reported adverse effects.(30-41) 
Finally, as outlined in sections 7.4 and 7.5, every effort 
has been made to minimize any risks associated with 
the infusion of somatostatin and glucagon. 
- The study will infuse a stable isotope (deuterium) 
instead of a radioactive isotope (tritium) as a glucose 
tracer. 
- The PI has personally performed the hyperinsulinemic, 
euglycemic clamp over 50 times in human subjects 
with T2DM and a similar number of times in canine 
studies.  
2. The procedure presents experiences to participants that are 
reasonably commensurate with those inherent in their actual 
or expected medical situation. 
- 
Hypoglycemia is a fact of life for most patients with 
insulin-treated diabetes. The average patient with 
T1DM experiences two episodes of hypoglycemia per 
week.(87) Every effort will be made to prevent 

Protocol Version #:  10 
 
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Protocol Date: October 24, 2017 
hypoglycemia from occurring during the clamp study at 
all, however. 
3. The procedure is likely to yield generalizable knowledge about 
the participants’ disorder or condition which is of vital 
importance for the understanding or amelioration of the 
participants’ disorder or condition. 
- 
As previously discussed, the proposed research will 
provide very important information about the root 
cause of IR in T1DM and possibly in MODY2, which in 
turn contributes considerably to macrovascular disease 
mortality. 
4. Adequate provisions are made for soliciting assent of the 
children and permission of their parents or legal guardians as 
detailed in the present IRB application. 
 
The study team will discuss all procedures, risks, and benefits with potential study 
participants, as part of the consent/assent process. An IRB approved written informed 
consent/assent document will be required for participation in this study. It is understood 
that consent/assent is a process and not a discrete event.  A participant’s decision to 
withdraw consent/assent will be respected throughout the duration of each subject’s 
participation in this study. It is also understood that there may be as-yet unknown or 
unanticipated adverse effects of this study. The study team will continually monitor for 
these effects and consider altering the protocol as needed to ensure patient safety.  
Changes in the procedures of the study, as well as any change(s) in the risks and/or 
benefits will be presented to and discussed with the subjects upon approval from the 
IRB for implementation of such revision(s), and any IRB revised written consent will be 
signed, as appropriate.   
 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
 
An AE will be considered to be any untoward medical occurrence in a subject, not 
necessarily having a causal relationship with the study.  The PI will continually monitor 
for any AEs.   
 
AEs will be graded according to following scale: 
 
0 = No adverse events 
1 = Mild: no limitation of usual activities, did not require treatment 
2 = Moderate: some limitation of usual activities, resolved with or without treatment,  
3 = Severe: inability to carry out usual activities, requiring medical intervention 
4 = Life-threatening or disabling 
5 = Death 
 
Hypoglycemia and hyperglycemia will be reported as adverse events only in the case of 
requiring the assistance of others due to loss of consciousness or DKA.  These will be 
considered serious adverse events (SAEs). 
 

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Protocol Date: October 24, 2017 
A member of the study team will contact each subject on the afternoon following the 
clamp study.  Subjects who underwent a muscle biopsy will be asked to grade their pain 
on a 1-10 scale.  Subjects taking insulin will be asked to report their 24-hour post-study 
glucose log numbers.  The PI will continuously monitor these post-study data for 
hypoglycemia and alter the procedure for preventing hypoglycemia described in section 
6.2.2.4 if needed.   
 
Consistent with FDA guidelines, SAEs will be defined as any untoward medical 
occurrence that: 
- 
requires inpatient hospitalization 
- 
results in persistent or significant disability 
- 
is suspected to cause a congenital anomaly or birth defect in a subject's unborn 
child 
- 
is life-threatening 
- 
results in death 
- 
is considered to be an important medical event based on appropriate medical 
judgement (e.g. bronchospsasm requiring emergency department referral, 
seizures that might not result in hospitalization). 
 
Additionally, an SAE's relationship to the study procedures will be assessed and graded 
as either: not related, unlikely, possible, probable, or definite. 
 
Any AE will be assessed for whether or not it was an anticipated problem. In accordance 
with DHHS guidance and consistent with 45 CFR part 46, an "unanticipated problem" will 
include any incident, experience, or outcome that meets all of the following criteria: 
 
1. unexpected (in terms of nature, severity, or frequency) given  
a. the research procedures that are described in the protocol-related 
documents, including the IRB-approved research protocol and informed 
consent document; and  
b. the characteristics of the subject population being studied; related or 
possibly related to participation in the research; and 
2. suggests that the research places subjects or others at a greater risk of harm. 
 
3. related or possibly related to participation in the research; 
 
4. suggests that the research places subjects or others at a greater risk of harm 
than was previously known or recognized. 
 
All unanticipated, non-serious AEs and the study team’s response to the non-serious AE 
will be included in a report at the time of annual continuing review.  The PI will review 
the AEs and notify the Data Safety Monitor (DSM), Dr. Kevin Niswender, and the IRB of 
any changes needed to the protocol. If needed, appropriate changes will be made to the 
consent form.   
 
In accordance with IRB policy, any unanticipated SAE that is considered possibly related 
to participation in the study will be reported within 7 calendar days of the PI's 

Protocol Version #:  10 
 
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Protocol Date: October 24, 2017 
notification of the event to the IRB and DSM. The study team will continue to follow or 
obtain documentation of the resolution of any SAE.   
 
The annual summary of all unanticipated adverse events and any audit reports will be 
sent to the IRB at the time of continuing review. A copy of this report will also be sent to 
the NIH who currently funds the PI on a K12 grant (and in the future will fund on an 
individual K-grant) with the Research Performance Progress Report.   
 
Data and safety monitoring activities for this study will continue until all subjects have 
completed their participation and until a sufficient amount of time has passed beyond 
which any study-related AEs are unlikely.   
 
This protocol will be reviewed annually (at a minimum) by the Vanderbilt IRB.  The goal 
of this process is to determine the risks and benefits of the study in the actual 
experience of subjects and that measures taken to minimize risks are adequate. 
 
9.0 
Study Withdrawal/Discontinuation 
 
Subjects will be free to withdraw from the study at any time, which will be made clear at 
enrollment. Subjects will be withdrawn from the study if:  
- 
Pregnancy is detected 
- 
The PI’s medical judgement is that participation places the subject at risk for 
harm 
 
10.0 Statistical Considerations 
 
 
- 
Sample size: The sample size in this cross-sectional study design (10 per control  
group, 16 per T1DM group, and 10 per MODY2 group) was calculated to detect a 
40% difference in mean glucose update (Rd) between MODY2 and T1DM subjects 
during maximal insulin stimulation (sample period 2) with an α-level of 5% and 
80% statistical power. The Rd variance and Rd for well controlled T1DM subjects 
utilized in sample size calculations were taken from Bergmann et al. where a 55% 
difference in Rd was seen between T1DM and control groups.(10)  
 
- 
Primary outcome: Differences in Rd during maximal insulin stimulation (sample 
period #2) between MODY2 and T1DM subjects will be evaluated using the Mann-
Whitney U test, presuming a non-parametric distribution in the data. Differences in 
Rd between MODY2 and control subjects and between T1DM and control subjects 
will be similarly determined. 
 
- 
Secondary outcome: The differences in suppression of glucose production (ΔRa), 
suppression of NEFA, and suppression of glycerol between each of the 3 subject 
groups will be assessed using the Mann-Whitney U test as for the primary 
outcome. 
 
- 
Timeline and interim analysis: I plan to study 18 subjects in year 1 and 12 
additional subjects in a second year of funding. One interim assessment of the 

Protocol Version #:  10 
 
29 
Protocol Date: October 24, 2017 
primary outcome (Rd) will be made after 5 studies per group before the end of the 
year 1 funding period. Obrien-Fleming bounds for a two-sided test with α-level of 
5% and a critical z-value of 2.80 (corresponding to a p-value of 0.0051) will be 
used to assess whether the study can be ended at interim analysis if there were a 
large difference in Rd between T1DM and MODY2 groups. At this point I will also 
analyze the variance in Rd and adjust the sample size if needed for subsequent 
studies in year 2 
 
11.0 Privacy/Confidentiality Issues 
 
A database will be designed for this study using REDCap (Research Electronic Data 
Capture) tools. REDCap is a secure, web-based application designed to support data 
capture for research studies, providing validated data entry, audit trails, seamless data 
downloads to common statistical packages, and mechanisms for importing data from 
external sources. It will reside on a secure server with access provided exclusively to the 
research personnel. Subjects will be identified with a study identification number. A key 
to the subject identification number will be kept in a separate locked file drawer to 
which only the Principal Investigator and research coordinators have access. Reports will 
thereby be generated without Protected Health Information (PHI) data, and access will 
be restricted so that statisticians, etc. don't have access to all data. 
 
Risk of leakage of PHI is minimized by keeping paper records in a locked cabinet and 
maintaining computerized records in the password protected REDCap data base. The 
principal investigator and the research staff are trained in HIPAA privacy regulations. 
The participant's identification is concealed, and a number is used as the identifier 
instead of the subject's name.  Only the principal investigator or members of the 
research team will have the list of study patient's names as the correlate with the study 
number.  
 
12.0 Follow-up and Record Retention 
 
The study is anticipated to last for two years.  The study results will be maintained 
indefinitely for research purposes.   
 
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Protocol Version #:  10 
 
38 
Protocol Date: October 24, 2017 
Appendix A: Pre-Clamp Glucose Log (image smaller to fit page) 
PRE-CLAMP GLUCOSE LOG
SUBJECT ID:
X
TOTAL CALORIES PER DAY:
2926
DATE
PRE-BG
TIME
CARBS (G)
CALORIES
PRE-BG
TIME
CARBS (G)
CALORIES
PRE-BG
TIME
CARBS (G)
CALORIES
BEDTIME-BG TIME
BEDTIME CARBS
CALORIES
POST-BG
TIME
INSULIN (U)
POST-BG TIME
INSULIN (U)
POST-BG TIME
INSULIN (U)
2 AM-BG
TIME
BEDTIME INSULIN
120
7:00 AM
50
150 12:00 PM
60
100
6:00 PM
90
150 10:00 PM
0
180
9:00 AM
5
200
2:00 PM
6
130
8:00 PM
9
120
2:00 AM
0
2 DAYS BEFORE
DAY BEFORE
ADMIT DAY
DATE
BG
TIME
CALORIES
TIME
BG
TIME
CALORIES
TIME
50 11:00 PM
1583
3:00 PM
814
11:00 PM
2 DAYS BEFORE
DAY BEFORE
ADMIT DAY
Abbreviations:
BG = Blood Glucose (blood sugar)
G = grams
U = units
Instructions to study participants:
For 3 days leading up to your overnight stay in the hospital ("ADMIT DAY") you have been asked to check your blood sugar (glucose) 8 times each 24 hour period.
With each of the 3 daily meals, please check your blood sugar before you begin eating and two hours after you begin eating.  Please check your sugar before going
to bed also.  If you have a bedtime snack, please check your blood sugar right before the bedtime snack.  Finally, please check your blood sugar at 2 AM.
These blood sugar numbers and the time they were checked should be written down in the table above.  At the top of the table, you will find an example of how
this should be done.  Please include your insulin doses and the grams of carbohydrates in each meal.
Please note the "total calories per day" on the top line above.  Your goal is to budget your food intake to try and match the number of calories you eat each day
to this number.  
If you need to check your blood sugar more often (if you have a low blood sugar, for example) please record the time and blood sugar number in the lower table.
If you have an additional snack, please note the calories in the lower table.  Remember, try to budget your eating to match the number of calories allowed per day.
NOTES
1219
1126
ADDITIONAL CALORIES & BG CHECKS
EXAMPLE
BEDTIME SNACK & OVERNIGHT
EXAMPLE
260
321
BREAKFAST
LUNCH
DINNER

Protocol Version #:  10 
 
39 
Protocol Date: October 24, 2017 
Appendix B: Post-Clamp Glucose Log (image smaller to fit page) 
POST-CLAMP GLUCOSE LOG
SUBJECT ID:
X
DATE
PRE-BG
TIME
PRE-BG
TIME
PRE-BG
TIME
BEDTIME-BG TIME
2 AM-BG
TIME
EXAMPLE
120
7:00 AM
150 12:00 PM
100
6:00 PM
150 10:00 PM
120
2:00 AM
CLAMP DAY
DAY AFTER 
HYPOGLYCEMIC EPISODES
DATE
TIME
BG
Abbreviations:
BG = Blood Glucose (blood sugar)
G = grams
U = units
Instructions to study participants: 
After going home, you should resume your routine medication regimen.
In the 24 hours following the completion of the clamp study, we would like for you to monitor closely for low blood sugar. 
After going home, at a minimum, please check your sugar at bedtime, at 2 AM, at breakfast, and at lunch. Please
check sugar before driving.  If your blood sugar is less than 120 mg/dL, eat an additional 15-30 grams of carbs
with 5-15 grams of protein.  As an example, one Luna bar has 26 grams of carbs and 9 grams of protein.
A package of 6 peanut butter and crackers has 24 grams of carbs and 5 grams of protein.
Please write your blood sugar numbers down using the tables above. We will call you approximately 24 hours after you have been discharged.
We will ask you to tell us these blood sugar numbers over the phone.  
INSULIN
0
0
60
6
9
90
INSULIN (U)
CARBS (G)
CARBS (G)
INSULIN (U)
BREAKFAST
LUNCH
COMMENTS
INSULIN (U)
50
5
BEDTIME SNACK & OVERNIGHT
DINNER
CARBS (G)
CARBS