A Study to Determine Iatrogenic Hyperinsulinemia's Contribution to Insulin Resistance and Endothelial Dysfunction in Type 1 Diabetes
Phase: NA
Conditions: Diabetes Mellitus, Type 1
Sponsor: Vanderbilt University Medical Center

Protocol Version #:6 
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Protocol Date:  July 29, 2021 
Determination of Iatrogenic Hyperinsulinemia’s 
Contribution to Insulin Resistance and 
Endothelial Dysfunction in Type 1 Diabetes 
Justin M. Gregory, M.D. 
Assistant Professor of Pediatrics 
Ian M. Burr Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics 
Vanderbilt University Medical Center 
Joshua A Beckman, M.D. 
Professor of Medicine 
Division of Cardiology, Department of Medicine 
Vanderbilt University Medical Center 
Cyndya A. Shibao, M.D. 
Associate Professor of Medicine 
Division of Clinical Pharmacology, Department of Medicine 
Vanderbilt University Medical Center 
Naji N. Abumrad, M.D. 
John L. Sawyers Professor of Surgery, 
Department of Sugery 
Vanderbilt University Medical Center 
Kevin D. Niswender, M.D., Ph.D. 
Associate Professor of Medicine 
Associate Professor of Medicine, Division of Diabetes, Endocrinology, and Metabolism 
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 
NCT04118374

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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 
 
Appendices 
Appendix 1: Overnight Insulin Infusion Protocol  
 

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1.0 Background 
Macrovascular disease is the leading cause of mortality in T1DM and is evident 
early in the condition’s progression. Despite numerous advances(1) to reduce 
hyperglycemia in T1DM over the past 25 years, coronary arterial disease (CAD) remains 
a profound contributor to excess mortality in T1DM.(2) 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.(3) Ischemic heart disease 
contributes the greatest percentage in estimated loss of life expectancy (36% in men, 
31% in women).(3) Even contemporary T1DM patients with glycemic control at target (i.e. 
HbA1c ≤ 6.9%) remain at a 3-fold increased risk from cardiovascular disease death.(4) 
The atherosclerotic changes of macrovascular disease begin in childhood and 
adolescence(5-9) and younger adults carry an extremely marked mortality risk.(10-13) 
Patients with disease onset before age 10 have a 30-fold higher risk for CAD above 
matched controls without T1DM.(14) Thus, pathophysiologic mechanisms that contribute 
to macrovascular disease appear early in the course of T1DM even when glycemic 
control is at target, making early therapeutic interventions necessary. 
Hyperinsulinemia is an unintended consequence of peripheral insulin delivery and 
may be the root cause of a significant portion of macrovascular disease burden in 
T1DM. Because peripheral insulin delivery bypasses first-pass hepatic extraction, from 
the moment therapy begins patients with T1DM will have basal insulin concentrations 
that are ≈2.5-fold higher in the peripheral circulation than matched controls with 
equivalent glycemia.(15-18) In large epidemiologic studies, hyperinsulinemia is a 
predictor for ischemic heart disease in nondiabetic individuals, independent of 
concomitant risk factors such as dyslipidemia and obesity.(19) This study will determine 
how much iatrogenic hyperinsulinemia is pathophysiologically linked with IR (Aim 1) and 
endothelial dysfunction (Aim 2) early in the course of T1DM. It will also test how much an 
intervention to lower hyperinsulinemia—a low carbohydrate diet—can improve IR and 
endothelial dysfunction (Aim 3).  
Iatrogenic hyperinsulinemia’s contribution to IR early in the course of T1DM is 
unestablished. IR is independently correlated with macrovascular disease in T1DM. (20-
22) 
My 
previous 
human 
subjects 
research 
strongly 
implicated 
iatrogenic 
hyperinsulinemia as the primary driver of IR in T1DM (see preliminary data). This work 
aligned with that of others who induced chronic hyperinsulinemia using differing 
approaches: overexpressing insulin gene expression the mouse(23, 24), delivering 
excess insulin into the hepatic portal vein of dogs(25), and infusing insulin into a 
peripheral vein of healthy humans for 2-3 days(26, 27). A common theme throughout 
these studies is that chronic hyperinsulinemia evokes a downregulation of insulin 
sensitivity. Although Yki-Järvinen et al. found insulin sensitivity improved during the 
partial clinical remission (PCR, a.k.a. “Honeymoon”) phase, the contribution of decreased 
iatrogenic hyperinsulinemia to the enhanced insulin sensitivity adjusted for other 
covariates has not been examined.(28) We will determine this relationship in Aim 1.  

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Iatrogenic hyperinsulinemia’s contribution to endothelial dyfsunction early in the 
course of T1DM is uncharacterized. Impaired endothelial function has been 
consistently observed in prepubertal,(29) midpubertal,(6) and postpubertal pediatric 
patients,(8) as well as young(30) and middle-aged(7) adults with T1DM. Several lines of 
research suggest insulin may exert beneficial vascular effects by increasing endothelial 
nitric oxide (NO) synthase gene expression(31) and activity(32, 33) and stimulating NO 
release and vasodilation.(34-36) On the other hand, hyperinsulinemia may exert 
detrimental vascular effects by amplifying mitogenic activity of growth factors, increasing 
expression of endothelial adhesion molecules, and enhancing monocyte adhesion to the 
endothelium.(34, 37, 38) Induction of hyperinsulinemia in healthy subjects to 
concentrations matching basal insulin levels typically seen in T1DM drastically diminished 
flow-mediated dilation (FMD), a frequently-used technique to quantify endothelial 
function.(39) Further study suggested this effect was mediated independent of insulin 
sensitivity.(34) Thus, my primary outcome in Aim 2 will determine how much clinically 
driven changes in peripherally delivered insulin doses modify brachial artery endothelial 
function during the early course of T1DM. As a secondary objective of Aim 2, I will use 
the contrast-enhanced ultrasound (CEUS) technique(40) to measure insulin-induced 
microvascular recruitment in adult participants.  
 
Therapies to lower hyperinsulinemia-mediated IR and endothelial dysfunction in 
T1DM are limited. To date, no study has attempted to improve IR or endothelial 
dysfunction by lowering the total daily dose of insulin (TDDinsulin). The dearth of these 
studies is partially related to concern that lowering TDDinsulin would worsen 
hyperglycemia. A diet that is lower in carbohydrates, however, is one approach to safely 
lower TDDinsulin without raising glycemia. Thus, in Aim 3, we will conduct a 2x2 crossover 
intervention study testing whether the reduction in TDDinsulin over a one-week low-
carbohydrate diet (LCD) intervention will result in improved insulin sensitivity compared 
with one-week standard carbohydrate diet (SCD).  
 
2.0 Rationale and Specific Aims 
Insulin resistance (IR) is consistently found among patients with type 1 diabetes (T1DM) 
and strongly links T1DM with atherosclerotic disease.(9, 20-22, 41-46) IR and nascent 
atherosclerosis, as characterized by endothelial dysfunction, are present early in 
T1DM.(6-9, 47) Coronary artery disease (CAD) is the leading cause of death in T1DM, 
(10, 13, 48) but the early contributors to the pathologic processes underlying this morbidity 
are not well-characterized.  
 
We have shown that IR in T1DM is minimally attributable to hyperglycemia, but is closely 
related to iatrogenic hyperinsulinemia. From the time of diagnosis, T1DM therapy relies 
on “unphysiologic” insulin delivery, where insulin injection into subcutaneous tissue rather 
than more physiologic delivery directly into the hepatic portal circulation results in 

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chronically elevated peripheral insulin levels. In the physiologic state, the liver clears ≈50% 
of secreted insulin before it reaches the peripheral circulation. As a result, hepatic insulin 
levels are normally ≈2-3 fold higher than insulin levels at peripheral tissues. By contrast, 
in T1DM injected insulin is directly absorbed into the peripheral circulation. Thus, patients 
with T1DM have basal insulin concentrations that are ≈2.5-fold higher in the peripheral 
circulation compared to non-diabetic individuals with matched glycemia.(49-51)  
 
Because 1) hyperinsulinemia is 
closely associated with IR (23-27, 
52), 2) exposure to iatrogenic 
hyperinsulinemia 
begins 
at 
diagnosis, and 3) hyperinsulinemia 
is an independent risk factor for CAD 
in the nondiabetic population,(19) 
the present proposal will test the 
hypothesis that iatrogenic-induced 
hyperinsulinemia 
independently 
correlates with IR and endothelial 
function early in the course of T1DM.  
 
To test this hypothesis, I will analyze the relationship (figure 1) between hyperinsulinemic 
exposure (quantified by average total daily dose of insulin, TDDinsulin) and insulin sensitivity 
(Aim 1) and early endothelial dysfunction (Aim 2) at multiple points in the progression of 
T1DM (figure 2). In a longitudinal study (T1DM SUBSTUDY in figure 2), I will study patients 
at three phases over the 12 months following diagnosis, initial diagnosis, honeymoon 
phase, and post-honeymoon phase. Each phase has distinct insulin exposure: 1) soon 
after diagnosis (STUDY1: TDDinsulin≈0.5 units/kg/day), 2) during partial clinical remission 
(PCR), a.k.a. “Honeymoon phase” (STUDY2: TDDinsulin<0.4 units/kg/day), and 3) after 
emergence from PCR (STUDY3: TDDinsulin>0.6 units/kg/day). Additionally, we will study 
healthy, matched, euglycemic control participants to assess the contribution of 
hyperinsulinemia to IR under four experimental conditions (CONTROL SUBSTUDY in 
figure 2): (i) endogenous insulin only (STUDY 4), (ii) overnight hyperinsulinemia (IV insulin 
infusion, STUDY 5), (iii) one week of mild hyperinsulinemia (glargine, STUDY 6), and (iv) 
both overnight insulin and one week of hyperinsulinemia (STUDY 7). To further test this 
hypothesis, I will conduct a 2x2 crossover intervention study in adults with T1DM testing 
whether the reduction in TDDinsulin associated with a one-week, isocaloric low-
carbohydrate diet (LCD) intervention will result in improved insulin sensitivity compared 
with one-week, isocaloric, standard carbohydrate diet (SCD) (STUDY B-C, figure 3). 
 
Iatrogenic
Hyperinsulinemia
(TDDinsulin)
Insulin resistance
(Clamp GIR)
1°: Large Artery 
Endothelial 
Dysfxn (%FMD)
2°: Insulin 
induced MVR 
(CEUS)
Aim 1
Figure 1: Correlations tested in Specific
Aims
GIR=glucose infusion rate; 1°= primary outcome; 2°= secondary 
outcome; %FMD=% change in flow-mediated vasodilation; 
MVR=microvascular recruitment
Aim 2
1°:Reduction in 
Hyperinsulinemia-
mediated insulin 
resistance
Aim 3

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quantified how much an intervention to lower hyperinsulinemia can improve IR. Thus, we 
will use a LCD as a means to lower hyperinsulinemia and multivariable linear regression 
analysis to quantify the reduction in IR adjusted for confounding covariables.  
 
 

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3.0 Animal Studies and Previous Human Studies 
My previous research aimed to determine the relative contributions of hyperglycemia and 
iatrogenic hyperinsulinemia to insulin resistance in T1DM. The study quantified tissue-
specific insulin sensitivity using a two-step, hyperinsulinemic, euglycemic, clamp in three 
cohorts (n=10/group) with differing insulinemia and glycemia: healthy controls (with 
euinsulinemia and euglycemia), MODY2 (with euinsulinemia and hyperglycemia), and 
T1DM (with hyperinsulinemia and hyperglycemia matching the MODY2 group). I assessed 
the relative contribution of hyperglycemia to insulin resistance by comparing insulin 
sensitivity between control and MODY2 groups. Likewise, the contribution of 
hyperinsulinemia was revealed by comparing insulin sensitivity between MODY2 and 
T1DM groups. 
 

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HbA1c for control, MODY2, and T1DM groups was 4.8 ± 0.4%, 6.2 ± 0.3%, and 6.6 ± 
0.5%, respectively. Each group had similar characteristics for additional factors that could 
influence insulin sensitivity (e.g. age, BMI, etc.). On the night prior to the clamp study, all 
participants began fasting and glucose was monitored overnight. T1DM subjects 
additionally discontinued their home insulin regimen and received a variable IV insulin 
infusion to achieve a plasma glucose concentration between 90-120 mg/dL by morning. 
Whereas fasting plasma insulin concentrations in the MODY2 and control groups were 
virtually the same, peripheral insulin delivery in the T1DM group resulted in a ≈2.5-fold 
higher fasting insulin level (figure 4A) to reach fasting plasma glucose concentrations 
similar to the other groups (figure 4B). In the first step of the clamp, we infused insulin at 
12 mU/m2/min, a rate chosen to partially suppress glucose production (Ra) and lipolysis. 
As figure 4C suggests, all groups saw a very similar degree of Ra suppression during first-
step insulin infusion. Because the majority of fasting glucose Ra is hepatic in origin (>90%), 
this finding suggested hepatic insulin sensitivity was no different between cohorts with 
their differing glycemic statuses. 
 
A different pattern was seen in peripheral tissues (fat and muscle), however. When we 
assessed insulin’s ability to suppress lipolysis by quantifying the decrease in non-esterified 
fatty acid (NEFA) during the first insulin infusion step, it was evident that control and 
MODY2 cohorts had nearly total NEFA suppression while a much smaller decrease 
occurred in the T1DM group (figure 4D). Likewise, when muscle tissue was stimulated by 
a high insulin infusion (40 mU/m2/min) in the second step, control and MODY2 cohorts 
had similarly large increases in glucose disposal (Rd) above baseline, while a smaller 
increase was seen in T1DM (figure 4E). Multivariable linear regression showed 
insulinemia, but not glycemia, was significantly associated with muscle insulin sensitivity.  

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4.0 Inclusion/Exclusion Criteria 
 
STUDY 1-7 (Aims 1-2) 
Table 1 lists inclusion and exclusion criteria for STUDY1-7 (Aims 1-2), which will be 
determined at an initial screening visit. If a complete metabolic panel (CMP) or complete 
blood count (CBC) was completed at Vanderbilt University Medical Center within 6 months 
prior the screening visit that lab study will be used in lieu of a new CMP or CBC.  
 
 
 
Rationale for inclusion and exclusion criteria: To minimize potential confounding effects of 
increased age,(53) intermittent ketoacidosis,(54) and increased BMI(55) on insulin 
sensitivity, the study will target relatively young patients with glycemic control within or 
near target and with BMIs at or below what would be expected in T1DM and the general 
population. Further, because patients who have diabetic ketoacidosis (DKA) at initial 
presentation are less likely to experience partial clinical remission (PCR) than patients 
who do not have DKA at presentation, DKA at diagnosis is an exclusion criterion for the 
longitudinal study.  
ALL GROUPS
Inclusion Criteria
- BMI: 18-28 kg/m2
Exclusion Criteria
- Severe hypoglycemia: ≥1 episode in the
past 3 months
- Diabetes comorbidities:
- Any
hospital
admissions
for
diabetic
ketoacidosis in the past 6 months.
- SBP > 140 mmHg and DBP > 100 mmHg
- eGFR
by
MDRD
equation
of
<
60
mL/min/1.73m2
- AST or ALT > 2.5 times the upper limit of
normal
- Hct < 35%
- Medications:
Any
antioxidant
vitamin
supplement (<2 weeks before a STUDY
visit),
any
systemic
glucocorticoid,
antipsychotic,
atenolol,
metoprolol,
propranolol, niacin, any thiazide diuretic,
any OCP with > 35 mcg ethinyl estradiol,
growth hormone, any immunosuppressant,
antihypertensive, any antihyperlipidemic
- Other: pregnancy, Tanner stage < 5, peri-
or post-menopausal women, active smoker
T1DM subjects
Inclusion criteria
- Age: 15-35
Exclusion criteria:
- Medications:
any
diabetes
medication
except insulin
Control subjects
Inclusion criteria
- Age: 18-35
- HbA1c: <5.5%
Table 1: Study Inclusion and Exclusion Criteria,
STUDY 1-7 (Aims 1-2)

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STUDY B-C (Aim 3) 
Table 2 lists inclusion and exclusion criteria for STUDY B-C (Aim 3), which will be 
determined at an initial screening visit.  
 
 
 
Rationale for inclusion and exclusion criteria: To minimize potential confounding effects of 
intermittent ketoacidosis (54) and increased BMI(55) on insulin sensitivity, the study will 
target with glycemic control within or near target and with BMIs at or below what would be 
expected in T1DM and the general population. Since the crossover design minimizes 
between-intervention variance, inclusion criteria for age are loosened compared with the 
criteria for STUDY 1-7. Additionally, because the total daily dose of insulin (TDDinsulin) is 
the key independent variable of interest in the study, each T1DM participant must use an 
insulin pump. An insulin pump digitally records all insulin a participant uses. Because the 
importance of glycemic variability in the data analysis, all T1DM participants must use a 
INCLUSION CRITERIA
- Age: 18-60
- HbA1c: 5.6-9.0%
- Insulin delivery: must use an insulin pump
- Glucose monitor: must use a CGM.
- BMI: 18-33 kg/m2
- Body mass: ≥50 kg
EXCLUSION CRITERIA
- Severe hypoglycemia: ≥1 episode in the
past 3 months
- Diabetes comorbidities:
- Any
hospital
admissions
for
diabetic
ketoacidosis in the past 6 months.
- SBP > 140 mmHg and DBP > 100 mmHg
- eGFR
by
MDRD
equation
of
<
60
mL/min/1.73m2
- AST or ALT > 2.5 times the upper limit of
normal
- Hct < 35%
- Medications:
Any
diabetes
medication
besides
insulin,
antioxidant
vitamin
supplement (<2 weeks before a STUDY
visit),
any
systemic
glucocorticoid,
antipsychotic,
atenolol,
metoprolol,
propranolol, niacin, any thiazide diuretic,
any OCP with > 35 mcg ethinyl estradiol,
growth hormone, any immunosuppressant,
antihypertensive, any antihyperlipidemic
- Other: pregnancy, Tanner stage < 5, peri-
or
post-menopausal
women,
active
smoker, gluten-free diet requirement
Table 2: Study Inclusion and 
Exclusion Criteria,
STUDY B-C (Aim 3)

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continuous glucose monitor. Finally, STUDY B-C will focus on recruiting adult participants, 
to avoid excessive recruiting overlap with STUDY1-7.  
 
5.0 Enrollment/Randomization 
 
5.1 Recruitment 
5.1.1 Recruitment Resources 
We will use multiple tools to identify prospective study participants.  The PI 
will contact intramural and extramural endocrinology colleagues at 
Vanderbilt and in the region to discuss this study and ask them to refer 
potential subjects. The Vanderbilt Eskind Diabetes Clinic is a large 
academic center that sees approximately 4,000 patients with T1DM 
annually. The study team will also use additional research tools including 
Research match, Subject locator, and My Research at Vanderbilt.  We will 
post flyers and send an institution-wide mass email through the Research 
Notifications Distribution List. In addition, we will post our research flyer on 
social media platforms (e.g. Facebook and Twitter) to target further 
potential T1DM and control group participants. For example, the Middle TN 
JDRF Chapter Facebook page has a large number of followers across the 
state of Tennessee many of whom may participate in our research.  
5.1.2 Initial Contact with Potential Participants 
Once the research team becomes aware of a potential subject, we will 
either telephone the participant (or parents in the case of potential pediatric 
participants) or visit with them in person during an endocrinology clinic visit. 
The initial discussion will aim to make a preliminary 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 or that the individual is interested in being a 
control subject 
- 
age 
- 
weight 
- 
height 
- 
most recent HbA1c (if applicable) 
- 
diabetes duration (if applicable),  
- 
medications 
- 
history of severe hypoglycemia in the past 3 months 
- 
history of any episodes of DKA in the past 6 months 
- 
physical activity level 

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- 
food preferences and restrictions 
Inclusion and exclusion criteria for STUDY1-7 AND STUDY B-C are listed 
in tables 1 and 2, respectively. We will invite eligible subjects to participate 
in the initial screening visit. The initial screening visit will occur either in the 
Vanderbilt Clinical Research Center (CRC) or in the Vanderbilt Eskind 
Diabetes Center. The participant will receive an electronic or paper copy of 
the informed consent document to review prior to the initial screening visit.  
5.2 Enrollment 
A member of the study team (i.e. key study personnel, KSP) will obtain written, 
informed consent or adolescent ascent with parental consent at the beginning of 
the screening visit as detailed in section 6.1.1.1. Once consent/assent is obtained, 
the participant will be enrolled in the study. A detailed description of special 
protection for adolescents as research subjects follows in section 7.3 below. 
 
The Vanderbilt CRC is located on the 2rd floor of Medical Center North on the 
Vanderbilt University Medical Center (VUMC) campus (1211 Medical Center Dr, 
Nashville, TN 37232; telephone number: 
 The Vanderbilt Eskind 
Diabetes Center is on the 8th floor of Medical Center East on the VUMC campus 
(1215 21st Ave S 8th floor, Nashville, TN 37232; telephone number: 
  
5.3 Randomization 
STUDY1-7 (Aims 1-2), include a longitudinal study of newly diagnosed T1DM 
patients during the first twelve months after diagnosis (T1DM SUBSTUDY, 
STUDY1-3) Because of the longitudinal nature of the T1DM substudy, no 
randomization process is needed for STUDY 1-3. The CONTROL SUBSTUDY 
investigates the effect of small doses of insulin in healthy, nondiabetic individuals 
over differing durations: one week (STUDY 5), one night (STUDY 6), both one 
week and one night (STUDY 7), and with no exogenous insulin at all (STUDY 4). 
Control participants will go through STUDY 4-7 in a random order. The study team 
will randomize participant into a treatment order group using a web-based 
randomization tool (http://www.sealedenvelope.com), a Microsoft Excel macro, or 
a randomization tool within Redcap. 
 
STUDY B-C (Aim 3), is a random-order, 2x2 crossover study. On one arm, 
participants will consume a low-carbohydrate diet (LCD). On the other arm, 
participants will consume a standard carbohydrate diet (SCD). As shown in figure 
3, T1DM and control participants will be randomized into one of two treatment 
orders (“A” vs. “B”).  
 

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0.6 units/kg/day, which is a common dosing range during the first 2-3 
months after diagnosis.  
- 
For participants to enter STUDY2, their mean TDDinsulin must be ≤ 0.4 
units/kg/day. This usually occurs between 3-6 months post-diagnosis.  
- 
To enter STUDY3, the TDDinsulin must exceed or equal 0.6 units/kg/day, 
which usually occurs by 12 months post-diagnosis. 
2. Participants selected for the CONTROL SUBSTUDY (n=20) will not have 
T1DM and have no chronic illnesses. They will participate in STUDY 4-7 (each 
CRC study visit follows the same protocol).  
 
6.1.1 Initial Screening Visit 
 
Screening visits will take place in either the Vanderbilt CRC or a room 
designated for research in the Eskind Diabetes Center, based on the 
convenience of the participant (e.g. they are already coming to a diabetes 
clinic visit) and availability of the room in the Eskind Diabetes Center.  
 
The purpose of the screening visit is to obtain informed, written 
consent/assent, conduct a history and physical exam, instruct the 
participant in the use of a continuous glucose monitor and a digital insulin 
pen device, calculate the estimated energy requirement to be consumed 
before STUDY1-7 visits, and show participants how to log their food intake 
on their digital device. 
 
Each screening visit will consist of the following: 
6.1.1.1 Consent/Assent 
The PI or designated KSP will obtain consent/assent from all 
participants. Consent/assent will be obtained in a private room in 
the CRC or Eskind Diabetes Center 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 in detail and provide time for discussing any 
questions. The study team will provide a copy of the consent/assent 
form to the participant and parents if applicable.  
6.1.1.2 History and Physical Exam 
The PI or designee will review each subject’s clinical history, 
perform a physical exam, and take anthropometric measurements. 
6.1.1.3 Blood draw and urine collection 

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If the participant has not had a complete metabolic panel (CMP), 
complete blood count (CBC) drawn at Vanderbilt University Medical 
Center within six months prior to the screening visit, blood will be 
drawn to determine eGFR, LFTs, and hematocrit meet inclusion 
and exclusion criteria for study. If a CMP or CBC was drawn within 
six months prior to the screening, those results can be used at the 
discretion of the PI. HbA1c will be drawn at the screening visit. 
Female participants will provide a urine sample to exclude 
pregnancy.  
6.1.1.4 Continuous Glucose Monitor Instructions 
The study team will instruct T1DM participants in the use of a 
continuous glucose monitor (CGM) such as Dexcom G6 or 
Medtronic Guardian for the monitoring of glycemia during the study. 
The team will then show T1DM participants how to upload both 
CGM data and insulin pump data (if using an insulin pump) into a 
HIPAA and FDA-compliant cloud-based, data-integration platform 
such as Dexcom Clarity, Medtronic Carelink, or Tidepool.  
 
Although the insulin glargine dose taken by nondiabetic participants 
in the CONTROL SUBSTUDY before STUDY 6 and 7 is low (0.2 
units/kg/day), this medication incrementally increases the risk of 
iatrogenic hypoglycemia. Accordingly, control participants will 
monitor their glucose at home during the week prior to each of the 
STUDY 4-7 visits. The PI may elect to use either CGMs or 
conventional capillary blood glucose monitors on control 
participants. As part of the initial screening visit, KSP will teach 
CONTROL SUBSTUDY to identify symptoms of hypoglycemia (e.g. 
shakiness, weakness, etc.), to check their glucose to confirm 
hypoglycemia (glucose < 70 mg/dL), and to use the commonly-
taught “rule of 15” to treat a hypoglycemic episode. If a conventional 
capillary glucose monitor is used, KSP will instruct CONTROL 
SUBSTUDY participants to check their glucose each morning prior 
to eating and each time he or she has symptoms of hypoglycemia. 
If a CGM is used, the team will show the control participants how to 
use the CGM device. CONTROL SUBSTUDY participants using a 
CGM will wear the device for 7-10 days prior to each STUDY 4-7 
visit.  
 
6.1.1.5 Digital Insulin Pen Instructions for T1DM SUBSTUDY participants 
The study team will provide a digital insulin pen such as the InPen 
(Companion Medical) or Echo (Novo Nordisk) to subjects with 

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T1DM who do have an insulin pump. Digital insulin pens use 
removable insulin cartridges and record each dose of insulin. The 
study team will show participants how to upload their insulin pen 
data using their HIPAA-compliant, FDA-approved smart phone app. 
6.1.1.6 Basal insulin instructions for CONTROL SUBSTUDY participants 
CONTROL SUBSTUDY participants (who do not have diabetes) 
will inject low doses of subcutaneous insulin glargine (0.2 u/kg/day) 
over 7 days preceding STUDY 6 and 7 and placebo over 7 days 
preceding STUDY 4 and 5. The study team will show these 
participants how to properly perform these injections at home. 
Please see section 7 regarding risk mitigation measures to prevent 
serious hypoglycemia.  
6.1.1.7 Diet instructions 
During the week preceding each STUDY visit, research participants 
will consume a weight-maintaining diet designed by the research 
team. The research team will discuss food preferences with each 
participant to design diet plan with standard macronutrient contents. 
The Mifflin-St. Jeor formula (57) will be used to calculate the 
estimated energy requirement (EER).  
EER for males (kcal/day) = PA (10 x weight [kg] + 6.25 x height [cm] 
– 5 x age [y] + 5) 
EER for females (kcal/day) = PA (10 x weight (kg) + 6.25 x height 
(cm) – 5 x age (y) – 161) 
Where PA is the physical activity coefficient:  
PA = 1.20 for little-to-no exercise 
PA = 1.38 for exercising 3 times weekly  
PA = 1.42 for exercising 4 times weekly  
PA = 1.46 for exercising 5 times weekly 
PA = 1.55 for daily exercise 
PA = 1.72 for daily intense exercise or exercising twice a day 
every day 
PA = 1.90 for daily exercise plus physical job 
Where exercise is 15-30 mins of elevated heart rate and intense 
exercise is 45+ mins of elevated heart rate. 
Macronutrient content will reflect the acceptable macronutrient 
distribution range of the Institute of Medicine’s Recommendations 
for Nutrition: carbohydrate (45%–65% of energy), protein (10%–
35% of energy), and fat (20%–35% of energy).(58)   

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Protocol Date:  July 29, 2021 
 
The study team will use the Nutrihand and/or the MyFitnessPal 
software program to design meal plans and participants will log their 
daily caloric intake. 
Nutrihand uses web-based or Smartphone app interfaces.  
Nutrihand creates a unique encrypted key whenever a healthcare 
professional adds a client to their account. That key can be used 
only once and by the specific email address used when the account 
was created. The participant will receive an automated email 
instructing them to log into the application for the first time. When 
he/she logs in, the first screen will ask the participant to agree to let 
the study team view their information and work with them. The 
participant can break the linkage with the study team at any time by 
removing the HIPAA key from their account. 
MyFitnessPal is a smartphone app and website that tracks diet and 
exercise to determine optimal caloric intake and nutrients for the 
users' goals. Users can either scan the barcodes of various food 
items or manually add them in the database of over five million 
different foods. Users can export their nutrient and exercise log as 
a de-identified CSV file (an example is shown in Appendix 2). 
Participants will email this de-identified nutrition log to the study’s 
email address. KSP will then upload the CSV file into the RedCAP 
database for subsequent data analysis.  
6.1.2 Management Before and In Between STUDY 1-7 visits 
Each T1DM participant’s endocrinologist will manage his or her glycemic 
control and insulin doses before and between T1DM SUBSTUDY visits. 
The study team will review each T1DM SUBSTUDY study participant’s 
insulin doses on at least a monthly basis between study visits. 
Each CONTROL SUBSTUDY participant will take insulin glargine 0.2 
u/kg/dose each day for seven days preceding STUDY 6 and 7 and an equal 
volume of a placebo injection (saline) prior to STUDY 4 and 5. The 
participant will be blinded to whether they are taking placebo or insulin 
glargine.  
 
Although the study takes several measures to mitigate the possibility of 
hypoglycemia in these participants (section 7.1), the investigator will not be 
blinded to whether or not control participants are taking insulin prior to 
CONTROL SUBSTUDY participants. This enables the investigator to 
rapidly alter the course of the intervention in the event of hypoglycemia.  
 

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Protocol Date:  July 29, 2021 
 
- 
Refrain from smoking and avoid secondhand smoke 
exposure for 12 hours 
- 
Consume no caffeine for 12 hours 
- 
Abstain from exercise for 12 hours 
 
Study participants will arrive to the CRC between 1700-2100 on the 
evening prior to STUDY visits.  
CRC staff will draw the following labs prior to conducting the clamp 
study: 
- 
Pregnancy test (urine or blood) on female participants to 
rule out pregnancy.  
- 
I-STAT potassium: to screen for hypokalemia 
 
T1DM participants who use an insulin pump will discontinue and 
detach their insulin pump at 2130 on the night of CRC admission. 
T1DM participants who take multiple daily insulin injections take 
their last basal insulin analog dose on the evening prior CRC 
admission and hold their basal insulin dose on the evening of 
admission. CRC staff or KSP will place an intravenous angiocathers 
in each arm.  
6.1.3.2 Overnight insulin infusion (T1DM SUBSTUDY participants only) 
At 2200 a continuous, IV infusion of regular human insulin 
(recombinant DNA origin) will be given to T1DM participants 
according to the protocol of Goldberg et al.(59), modified for use in 
healthy patients with T1DM (Appendix 1). This protocol targets a 
glucose of 90-120 mg/dL by the next morning. CRC staff will check 
blood glucose of T1DM participants on an hourly basis using a 
calibrated, hospital bedside glucose monitor. This protocol was 
implemented in a previous study (IRB # 161504) and achieved ideal 
glycemic control as shown in the figure below (n=9 T1DM 
participants, figure shows means ± SD for glucose and means for 
insulin infusion rate). 

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Protocol Date:  July 29, 2021 
 
 
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 experiences 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 PI will postpone 
the following morning’s clamp study to a later date because of the 
confounding influence of the counter-regulatory response to 
hypoglycemia.   
The PI will be readily available to CRC nursing staff via telephone 
and 
  
6.1.3.3 Overnight IV saline infusion (CONTROL SUBSTUDY 4 and 6) 
Non-diabetic control subjects participating in the CONTROL 
SUBSTUDY will receive an IV infusion of isotonic saline at a rate of 
sufficient to keep the vein patent. Blood glucose will be checked 
every 1-2 hours overnight.  
6.1.3.4 Overnight IV insulin fusion (CONTROL SUBSTUDY 5 and 7) 
Non-diabetic control subjects will receive an IV infusion of regular 
human insulin at a rate of 8 mU/m2/min. Blood glucose will be 
checked every hour overnight. If the blood glucose level falls to 
below 75 mg/dL, IV glucose will be infused to maintain blood 
glucose between 75 and 100 mg/dL.  
6.1.3.5 Contrast-enhanced ultrasound (CEUS) 
Beginning at approximately 6 AM, baseline vital signs will be 
measured. We will then measure microvascular blood volume 

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Protocol Date:  July 29, 2021 
 
(MBV) using CEUS twice as shown in figure 6. The study team will 
measure MBV during basal conditions (CEUS-0) and insulin 
stimulated conditions (CEUS-1). Because perflutren is not FDA 
approved for use in the pediatric population, at present we will only 
use this technique in adult participants.  
A secondary objective of the study is to determine the relationship 
between hyperinsulinemia and the magnitude of insulin-induced 
microvascular recruitment (MVR, i.e. MBV1-MVB0). The CEUS 
technique employed is modeled after the protocol of Sjøberg et 
al.(40)  
We will fix a linear-array transducer connected to an ultrasound 
system to the forearm, allowing for cross-sectional imaging of the 
forearm muscle bed. 
Perflutren lipid microspheres (Definity, Lantheus Medical Imaging) 
will be activated by a vial mixer at 4,500 oscillations per minute for 
45 seconds. Perflutren lipid microspheres in suspension in a 1.5 mL 
will be diluted at bedside to 20 mL with isotonic saline in a sterile 
fashion. The diluted microsphere solution will then be infused 
intravenously at 1.2-1.5 mL/min using a rotating syringe pump to 
ensure a homogenous microsphere solution. After beginning the 
perflutren lipid microsphere infusion, there will be a 10-minute 
equilibrium period to allow the acoustic intensity signal to reach a 
steady plateau before measuring MBV. A region of interest (ROI) 
will be marked on a cross-section of the forearm avoiding areas of 
connective tissues or large blood vessels. The same ROI will be 
used in subsequent MBV measurements.  
Real-time imaging will be conducted using a low ultrasound 
mechanical index of 0.08. At this mechanical index, microspheres 
resonate without destruction. Thus, the acoustic index, generated 
by the resonating bubbles, is proportional to the microsphere 
concentration (and by extension blood volume) in the ROI interest.  
Once the 10-minute equilibrium period is complete, a high (1.20) 
mechanical index pulse will be delivered by the transducer to 
completely 
destroy 
the 
microspheres. 
This 
allows 
the 
replenishment of the microspheres within the microvasculature 
inside the ultrasound beam’s ROI to be measured. Following the 
high mechanical index pulse, the microspheres will be replenished 
within the microvasculature and the acoustic intensity across the 
ROI will be recorded. These data will then be exported to 
quantification software to determine MBV, calculated in accordance 
with Wei et al.(60)  
6.1.3.6 Flow-mediated dilation (FMD) 

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Protocol Date:  July 29, 2021 
 
After CEUS-0, we will determine FMD. Originally developed in 
1992, the FMD technique has become the most commonly utilized 
non-invasive 
method 
for 
assessing 
vascular 
endothelial 
dysfunction.(61) The study team will use the Phillips EPIQ 7C 
ultrasound machine with an L12-3 transducer. Ms. JoAnn Gottlieb, 
an ultrasonographer with extensive experience using this 
technique, will assist in these studies.  
Baseline Measurements: The baseline radial artery diameter and 
velocity will be measured. The brachial artery will be scanned 
beginning at the insertion of the biceps and proceeding proximally. 
Color flow imaging will verify the brachial artery and to locate 
collateral vessels to serve as landmarks in sequent studies. Once 
a suitable position is found, brachial artery diameter will be 
measured over at least 10 cardiac cycles. Velocity will be averaged 
over at least a 10-20 second period. 
Vascular occlusion: A pillow will support each participant’s arm with 
the palm facing forward. We will position an appropriately sized 
blood pressure cuff 5 cm distal to the elbow. A rapid cuff inflator will 
to occlude radial artery blood flow. The cuff will be inflated to at least 
25-50 mm Hg above systolic blood pressure for five minutes to elicit 
a reactive hyperemic stimulus for endothelium mediated 
vasodilation. 
Reactive Hyperemia (Post-cuff release) Measurements: Post-cuff 
measurements of arterial diameters and blood velocities will be 
initiated ten seconds prior to cuff release and continued for three 
minutes following cuff release.  
FMD Analysis: The primary outcome of the FMD study is %FMD, 
defined as: 
%𝐹𝐹𝐹𝐹𝐹𝐹= 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑ℎ𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦−𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏
𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏
 
6.1.3.7 Endothelium independent vasodilation: After the participant rests 
for another 10 min, we will measure nitroglycerin-induced 
vasodilation as an index of endothelium-independent vasodilation. 
We will administer a 400 μg sublingual dose of nitroglycerin and 
assess the brachial artery response by imaging the artery 
continuously for 3 min. We will quantify endothelium-independent 
vasodilation as the percent increase in brachial artery diameter 3 
min after the nitroglycerin dose.  
6.1.3.8 Basal blood measurements 

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Protocol Date:  July 29, 2021 
 
Following FMD, we will draw blood levels of the hormones, 
metabolites, and cytokines listed in table 3 up to three times over a 
30 min baseline sampling period.  
Table 3: Labs drawn during BASAL period 
 
6.1.3.9 Hyperinsulinemic-euglycemic clamp (EUGLY CLAMP) 
We will conduct a 120 min hyperinsulinemic, euglycemic clamp 
after drawing basal labs. Regular human insulin will be infused into 
a peripheral angiocatheter at 40 mU/m2/min. Plasma glucose will 
be monitored every 5-10 minutes using a YSI Glucose Analyzer 
(Yellow Springs, OH), the gold standard instrument for glucose 
concentration analysis.  IV glucose will be infused to maintain 
plasma glucose at approximately 90-100 mg/dL throughout the 
study.   
6.1.3.10 
CLAMP sampling period (SAMPLE) 
A second sampling period will measure the blood parameters listed 
in table 3 under insulin-stimulated conditions during the last 30 
minutes of the clamp.  
The total blood volume from each study is calculated to equal 124 
mL.  
6.1.3.11 
DEXA scan 
Prior to discharge, 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). 
6.1.3.12 
STUDY visit completion 
Upon completion of the study, we will stop the insulin infusion and 
allow the study subject to eat. The study team will continue 
monitoring plasma glucose for at least an additional hour and the 
steadily decrease the glucose infusion rate (GIR) to maintain 

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Protocol Date:  July 29, 2021 
 
glucose > 120 mg/dL. We will instruct T1DM participants to resume 
their home regimen, however they will be instructed to check 
glucose before dinner, bedtime, and at 2 AM following the study.  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.  
6.1.3.13 
Rescheduling STUDY visit 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. In the event 
such a technical issue occurs, the PI may elect to reschedule a new, 
repeat STUDY visit provided he deems the technical issue poses 
no serious danger to the participant were it to occur again and the 
participant wishes to repeat the study.  
 
6.2 Research visits: STUDY B-C (Aim 3) 
Each subject will participate in three research visits to complete participation as 
depicted in figure 7. The first visit (“SCREEN” in figure 7C) will determine the 
potential participant’s eligibility and allow the study team to provide instructions to 
the participants. If the participant meets eligibility criteria he/she will begin a 1-2-
week “run-in” period within six weeks of the screening visit. After the run-in period, 
the participant will be randomized to consume either 1 week (+/- 1 day) of a 
standard carbohydrate diet (SCD) or 1 week (+/- 1 day) of consuming a low 
carbohydrate diet (LCD) as shown in figure 7. On the final day of the intervention, 
the participant will consume a standardized 2,000 kilocalorie SCD or LCD provided 
by the study team. After a week (+/- 1 day) of being on either intervention, 
participants will report to the CRC for the first of two “STUDY” visits, where insulin 
sensitivity and endothelial dysfunction will be assessed (STUDY B). Following 
STUDY B, participants will begin a washout period of approximately 3 weeks 
where they will resume their usual diabetes management. The study team will 
schedule STUDY C to follow STUDY B by no more than one month in male 
participants. For female participants, STUDY B and C will occur during the follicular 
stage of consecutive menstrual cycles (i.e., day 2-10 of the menstrual cycle). For 
1 week (+/- 1 day) preceding STUDY C, each participant’s intervention will cross-
over to either LCD or SCD. On the final day of the second intervention, the 
participant will again consume a standardized 2,000 kilocalorie SCD or LCD 
provided by the study team. Upon completion of the second 1-week (+/- 1 day) 

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Protocol Date:  July 29, 2021 
 
- 
obtain informed, written consent/assent,  
- 
conduct a history and physical exam,  
- 
confirm each participant is able to properly use their insulin pump and 
continuous glucose monitor (CGM),  
- 
calculate the estimated energy requirement to be consumed before 
during the study period,  
- 
obtain a blood sample to ensure inclusion/exclusion criteria are met, 
- 
provide the participants with their meal plans and provide standardized 
meals for the day prior to STUDY visits,  
- 
show participants how to log their food intake on their digital device, 
and 
- 
perform a DEXA scan to quantify fat-free mass 
 
Each screening visit will consist of the following: 
6.2.1.1 Consent 
The PI or designated KSP will obtain consent from all participants. 
Consent will be obtained in a private room in the CRC or Eskind 
Diabetes Center 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 forms with the participant 
in detail and provide time for discussing any questions. The study 
team will provide a copy of the consent form to the participant.  
6.2.1.2 History and Physical Exam 
The PI or designee will review each subject’s clinical history, 
perform a physical exam, and take anthropometric measurements. 
6.2.1.3 Continuous Glucose Monitor and Insulin Pump Instructions 
The study team will confirm T1DM participants are able to 
appropriately use their home continuous glucose monitors (CGMs) 
such as Dexcom G6 or Medtronic Guardian for the monitoring of 
glycemia during the study. The team will then show T1DM 
participants how to upload both CGM data and insulin pump data 
into a HIPAA and FDA-compliant cloud-based, data-integration 
platform such as Dexcom Clarity, Medtronic Carelink, or Tidepool. 
The team will then show T1DM participants how to upload both 
CGM data and insulin pump data into a HIPAA and FDA-compliant 
cloud-based, data-integration platform such as Dexcom Clarity, 
Medtronic Carelink, or Tidepool.  
 

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Protocol Date:  July 29, 2021 
 
6.2.1.4 Blood draw and urine collection 
If the participant has not had a complete metabolic panel drawn and 
a complete blood count at Vanderbilt University Medical Center 
within six months prior to the screening visit, blood will be drawn to 
determine whether the eGFR, AST, ALT, and hematocrit meet 
inclusion and exclusion criteria for study. If a complete metabolic 
panel was drawn within six months prior to the screening, those 
results can be used at the discretion of the PI.  
Female participants will provide a urine sample to exclude 
pregnancy.  
6.2.1.5 Diet instructions 
The research team will discuss food preferences with each 
participant to design a weight-maintaining diet plan. The Mifflin-St. 
Jeor formula (57) will be used to calculate the estimated energy 
requirement (EER).  
EER for males (kcal/day) = PA (10 x weight [kg] + 6.25 x height [cm] 
– 5 x age [y] + 5) 
EER for females (kcal/day) = PA (10 x weight (kg) + 6.25 x height 
(cm) – 5 x age (y) – 161) 
Where PA is the physical activity coefficient:  
PA = 1.20 for little-to-no exercise 
PA = 1.38 for exercising 3 times weekly  
PA = 1.42 for exercising 4 times weekly  
PA = 1.46 for exercising 5 times weekly 
PA = 1.55 for daily exercise 
PA = 1.72 for daily intense exercise or exercising twice a day 
every day 
PA = 1.90 for daily exercise plus physical job 
Where exercise is 15-30 mins of elevated heart rate and intense 
exercise is 45+ mins of elevated heart rate. 
 
The study team will devise two isocaloric diet plans for each 
participant. One plan, the standard carbohydrate diet (SCD), will 
reflect the acceptable macronutrient distribution range of the 
Institute 
of 
Medicine’s 
Recommendations 
for 
Nutrition: 
carbohydrate (45%–65% of energy), protein (10%–30% of energy), 
and fat (25%–35% of energy) (figure 7A).(58)  The other plan, the 
low carbohydrate diet (LCD), will reduce the percent caloric intake 

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Protocol Date:  July 29, 2021 
 
from carbohydrates and replace that amount with calories from 
dietary fat. Thus with the LCD, 45%–65% of energy will come from 
fat, 10-30% of energy will come from protein, and 25%–35% of 
energy will come from carbohydrates (figure 7B). The study team 
will provide standardized, 2,000 kcal/day diets for participants to 
consume on the day preceding STUDY B and C visits. The 
macronutrient content of these standardized meals will match the 
LCD or SCD intervention.  
 
As with STUDY1-5, the study team will use the Nutrihand and/or 
MyFitnessPal software program to design meal plans and 
participants will log their daily caloric intake.  
Nutrihand uses web-based or Smartphone app interfaces.  
Nutrihand creates a unique encrypted key whenever a healthcare 
professional adds a client to their account. That key can be used 
only once and by the specific email address used when the account 
was created. The participant will receive an automated email 
instructing them to log into the application for the first time. When 
he/she logs in, the first screen will ask the participant to agree to let 
the study team view their information and work with them. The 
participant can break the linkage with the study team at any time by 
removing the HIPAA key from their account. 
MyFitnessPal is a smartphone app and website that tracks diet and 
exercise to determine optimal caloric intake and nutrients for the 
users' goals. Users can either scan the barcodes of various food 
items or manually add them in the database of over five million 
different foods. Users can export their nutrient and exercise log as 
a de-identified CSV file (an example is shown in Appendix 2). 
Participants will email this de-identified nutrition log to the study’s 
email address. KSP will then upload the CSV file into the RedCAP 
database for subsequent data analysis.  
6.2.1.6 DEXA scan 
Lean body and fat mass will be measured using dual-energy X-ray 
absorptiometry (DEXA) (Lunar Prodigy, enCore software version 
10.5, GE Medical Systems). 
 
6.2.2 Management Before and In Between STUDY B-C visits 
Each T1DM participant’s will manage his or her glycemic control before and 
between STUDY B-C visits using insulin as prescribed by their 
endocrinologist. The study team will monitor adherence by reviewing each 
participant’s dietary log, CGM data, and insulin pump data in an ongoing 

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Protocol Date:  July 29, 2021 
 
usual treatment routine (e.g., consuming 15 grams of 
carbohydrates).  
Insulin delivery: Participants using an insulin other than insulin 
lispro will be provided with this medication for use during the 24-48 
hours that precede each STUDY visit, thereby standardizing insulin 
delivery during the 24-48 hours prior to STUDY B and C. 
The inclusion criteria stipulate participants must already use an 
insulin pump and continuous glucose monitor. Based on which 
pump the participant uses, these patients can be further 
categorized by those individuals receiving insulin via hybrid closed 
loop (HCL) technology and those receiving insulin using sensor-
augmented pump therapy. On the night prior to each study visit, 
individuals will receive insulin based on which modality the 
participant uses. 
- 
Participants using HCL technology for their routine insulin 
regimen (e.g., those using the Tandem T:Slim pump and 
Medtronic pumps connected to a Dexcom CGM) will 
continue 
using 
their 
insulin 
pumps 
per 
their 
routine.Participants using sensor-augmented pump therapy 
will continue their routine treatment with the addition that the 
study team will ask them to give a corrective dose of insulin 
as needed for hyperglycemia using their usual insulin dose 
at bedtime. 
 
Study participants will arrive to the CRC at approximately 0700 on 
the morning of each STUDY B-C visit. Weight and height will be 
measured. A urine pregnancy test will be drawn on female 
participants to rule out pregnancy.  
 
 
6.2.3.2 Flow-mediated dilation (FMD) 
 
 
Study staff will instruct each participant to lie comfortably prone in 
bed. After approximately ten minutes of resting, we will determine 
FMD. The approach for measuring FMD in STUDY B-C is the same 
as that described for STUDY 1-5 in section 6.1.3.4. 
6.2.3.3 Basal blood measurements 
Following FMD, an intravenous angiocather will be placed in each 
arm; one used for hormone and dextrose infusion and the other 

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Protocol Date:  July 29, 2021 
 
used for sampling blood glucose. An I-STAT machine will be used 
to screen for hypokalemia upon obtaining IV access. We will draw 
blood levels of the hormones, metabolites, and cytokines listed in 
table 4 up to three times over a 30 min baseline sampling period.  
 
 

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Protocol Date:  July 29, 2021 
 
Table 4: Labs drawn during BASAL period 
 
6.2.3.4 Hyperinsulinemic-euglycemic clamp (EUGLY CLAMP) 
In STUDY B-C, we will conduct a 150 min hyperinsulinemic, 
euglycemic clamp after drawing basal labs in the same manner 
described for STUDY 1-5 in section 6.1.3.8.  
6.2.3.5 CLAMP sampling period (SAMPLE) 
A second sampling period will measure the blood parameters listed 
in table 4 under insulin-stimulated conditions up to three times 
during the last 30 minutes of the clamp.  
The total volume of all blood drawn from each STUDY B-C visit is 
calculated to equal 127 mL. 
6.2.3.6 STUDY visit completion 
Upon completion of the study, we will stop the insulin infusion and 
allow the study subject to eat. The study team will continue 
monitoring plasma glucose for at least an additional hour and then 
steadily decrease the glucose infusion rate (GIR) to maintain 
glucose > 120 mg/dL. We will instruct T1DM participants to resume 
their home regimen, however they will be instructed to check 
glucose before dinner, bedtime, and at 2 AM following the study.  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.  
6.2.3.7 Rescheduling STUDY visit 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 

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Protocol Date:  July 29, 2021 
 
hypoglycemia (glucose < 55 mg/dL) may occur for those 
participants receiving an overnight IV insulin infusion. In the event 
such a technical issue occurs, the PI may elect to reschedule a new, 
repeat STUDY visit provided he deems the technical issue poses 
no serious danger to the participant were it to occur again and the 
participant wishes to repeat the study.  
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 (62, 63). 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 Hyperinsulinemia 
 
The hyperinsulinemic, euglycemic clamp exposes participants to high insulin 
levels, potentially leading to hypoglycemia and hypokalemia. As shown in figures 
6 and 8, the insulin infusion rate during the clamp will be 40 mU/m2/min. This 
infusion rate is anticipated to result in plasma insulin concentrations between 50 
and 130 μU/mL,(64, 65)  levels that are within the range of physiological 
hyperinsulinemia and comparable to concentrations seen after meal ingestion.(65-
67) By comparison, the most commonly used insulin infusion rate in the treatment 
of diabetic ketoacidosis is 0.1 U/kg/hr.(56) 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. 
 
Nondiabetic CONTROL SUBSTUDY participants will be exposed to low levels 
of iatrogenic hyperinsulinemia from low doses of insulin glargine (STUDY 6-7) 

Protocol Version #:6 
 
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Protocol Date:  July 29, 2021 
 
and overnight IV regular insulin infusions prior to STUDY visits (STUDY 5 and 
7). The insulin glargine dose of 0.2 u/kg/day was selected to induce long-term 
(7 days) iatrogenic hyperinsulinemia while avoiding the confounding effect of 
hypoglycemia. Based on a previous pharmacokinetic-pharmacodynamic study 
of glargine, this dose is anticipated to drop fasting plasma glucose by no more 
than 10 mg/dL.(68) Because insulin glargine is a long-acting insulin analog, we 
do not anticipate it will cause a rapid drop in plasma glucose concentrations, 
but rather a slow decrease over the course of hours. For short-term 
hyperinsulinemia, participants will receive intravenous (IV) regular insulin at 8 
mU/m2/min for 8 hours overnight preceding the STUDY visit. Based on my 
previous research, I anticipate this dose will raise fasting plasma insulin 
concentrations in CONTROL SUBSTUDY participants from ≈8 μU/mL to ≈21 
μU/mL, matching the average fasting insulin concentration of T1DM 
SUBSTUDY participants.(69) 
We will use the following safety provisions to minimize risk of hypoglycemia and 
hypokalemia: 
7.1.1 Glucose monitoring 
Glucose levels will be monitored every hour during the overnight insulin 
infusion for T1DM participants as outlined in Appendix 1.  Because 
CONTROL SUBSTUDY participants will receive IV insulin overnight prior 
to STUDY 5 and 7, we will monitor every hour in the CRC on the night 
preceding these studies. We will monitor plasma glucose concentrations 
every 5-10 minutes throughout the hyperinsulinemic clamp to guide the 
variable dextrose infusion and ensure euglycemia. Because of the frequent 
glucose monitoring, the study team can quickly detect a trend toward 
hypoglycemia before it actually occurs. If hypoglycemia does occur, 
however, this frequent monitoring allows for rapid detection of the low 
glucose concentration and rapid corrective treatment with an infusion of IV 
glucose (dextrose).  
7.1.2 Correction of potential hypoglycemia 
A licensed MD, NP, or PA will be on call throughout overnight CRC stay 
and at bedside during the clamp procedure. Concentrated dextrose will be 
at bedside to treat iatrogenic hypoglycemia. If hypoglycemia occurred, the 
study team could rapidly treat the low blood glucose.  For example, a 75 
mL IV bolus of 20% dextrose (15 gm of glucose) would bring glucose up 
above the hypoglycemic range in less than 5 minutes. CRC staff could 
deliver the dextrose into either of the two IV angiocatheters available during 
the study. 
7.1.3 Avoiding post-discharge hypoglycemia 
KSP will counsel subjects on the risk of late hypoglycemia the evening and 
night following the clamp. We will advise them of the importance of 

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checking their blood glucose at bedtime and during the night to maintain a 
minimum of 120 mg/dL.  
7.1.4 Monitoring CONTROL SUBSTUDY participants for hypoglycemia 
CONTROL SUBSTUDY participants will take 0.2 u/kg/day of long-acting 
insulin glargine during the week prior to STUDY 6-7 and matched saline  
placebo during the week prior to STUDY 4-5. As discussed at the beginning 
of section 7.1 this dose is expected to decrease the fasting plasma glucose 
concentration by 10 mg/dL or less. Even so, the possibility of hypoglycemia 
in these nondiabetic control participants cannot be entirely avoided. 
Accordingly, these participants will monitor their glucose closely during the 
week prior to STUDY visits 4-7.  The study will supply CONTROL 
SUBSTUDY participants with a conventional capillary blood glucose meter 
or a continuous glucose monitor (CGM). If a conventional capillary blood 
glucose meter is used, KSP will instruct participants to check their blood 
glucose each morning prior to breakfast (when they are most likely to have 
hypoglycemia after fasting overnight) and more often if needed at the 
discretion of the PI. If a CGM is used, the device will monitor glucose 
continuously during the week prior to STUDY visits 4-7. At the screening 
visit, KSP will teach CONTROL SUBSTUDY participants to identify 
symptoms of hypoglycemia. Should these symptoms occur, CONTROL 
SUBSTUDY participants will check their glucose level using either their 
conventional capillary glucose meter or their CGM. If the glucose is less 
than 70 mg/dL, participants will use the commonly-taught “Rule of 15” 
(https://www.diabetes.org/diabetes/medication-management/blood-
glucose-testing-and-control/hypoglycemia) to treat the hypoglycemia. 
Participants will contact the study team after such an event occurs. The PI 
will then consider altering the insulin glargine dose or even withdrawing the 
participant based on his clinical judgement, in conjunction with the data 
safety monitor. 
7.1.5 Safety provisions to minimize hypokalemic risk 
Hyperinsulinemia causes extracellular potassium to shift into the 
intracellular space, a factor that could contribute to apparent hypokalemia. 
Vital signs will be monitored throughout the STUDY visit procedure. 
Potassium will be monitored using bedside istat monitor. Oral potassium 
replacement will be given as needed at the discretion of the PI or his 
designated MD, NP, or PA KSP. 
7.2 Risks Associated with Perflutren Lipid Microsphere Infusion 
7.2.1 Clinical and Research Application of Perflutren 
Perflutren is an ultrasound contrast agent indicated for clinical use in 
patients with suboptimal echocardiograms to opacify the left ventricular 
chamber and improve the delineation of the left ventricular endocardial 

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STUDY visits, which include two infusions of perflutren lipid microspheres. 
Further, the PI will be under the supervision of a board-certified cardiologist 
(Dr. Beckman) and internist (Dr. Shibao) who have extensive experience 
using this technique. The PI will discuss the conduct of the study on a 
routine basis. In rare circumstances, the PI may elect to allow a designed 
KSP (MD, NP, or PA) to provide medical supervision in his absence. 
STUDY visits will be conducted in the Vanderbilt Clinical Research Center, 
a state-of-the-art facility that has cardiac monitoring and resuscitation 
equipment readily available. Vital signs will be monitored throughout each 
STUDY visit.  
7.3 Anemia 
For STUDY B-C we will collect a small blood sample (10 mL) at the screening visit. 
This blood sample will include a complete blood count (CBC) test to ensure each 
participant’s hematocrit is at least 35%.  
Within eight weeks of the screening visit, we will conduct two hyperglycemic, 
euglycemic clamps on STUDY B-C (Aim 3) participants, with up to one month  
separating each study (see section 6.2). The total amount of blood drawn in each 
STUDY B-C visit equals 127 mL. This includes the volume of fluid in the 
angiocatheter that must be cleared with each blood draw to ensure blood samples 
are not diluted (41 mL in total). This fluid, often called “waste,” is mostly saline 
running through the angiocatheter to ensure the catheter remains patent but 
contains some blood. Thus, the maximum amount of total blood drawn in each 
study is calculated to equal 127 mL. Thus, we calculate a maximum volume from 
the STUDY B-C visits of 254 mL over one month. 
As reviewed in a World Health Organization Bulletin,(78) while several guidelines 
are available for reference, there is a limited amount of direct evidence on which 
to base them. We have set 50 kg as the minimum required body mass STUDY B-
C participants to align with guidance from the Wayne State University Institutional 
Review Board for Minimal Risk blood collection which states: 
 
 “When the collection of blood samples presents minimal risk to adult subjects they 
may be one by finger stick, heel stick, ear stick, or venipuncture under the following 
conditions and guidelines: 
a. 
Blood samples may be collected for research purposes from healthy, 
nonpregnant adults who weigh at least 110 pounds. The amounts drawn may not 
exceed 550 ml in an 8 week period and collection may not occur more frequently 
than 2 times per week”.(79) 
 
Further, our approach aligns NIH policy M95-9(rev.) “Guidelines for Limits of Blood 
Drawn for Research Purposes in the Clinical Center.” For adult patients and 
volunteers, the policy states, “The amount of blood that may be drawn from adult 

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Protocol Date:  July 29, 2021 
 
patients and volunteers (i.e.,those persons 18 years of age or older) for research 
purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any 
eight week period.” 
 
STUDY 1-5 poses less risk for anemia. The total blood volume drawn in each 
STUDY 1-5 visit is 124 mL. Although longitudinal study (STUDY 1-3) participants 
are studied three times, each study visit is separated by more time. We do not 
anticipate the time between STUDY1 and STUDY2 will ever be less than 1 month 
and anticipate the normal interval between STUDY1 and STUDY2 will be between 
2-4 months.  
 
In addition to obtaining a complete blood count at screening, the PI will clinically 
monitor all volunteers for signs and symptoms of anemia throughout their 
participation. Specifically, the PI will monitor participants for easy fatigability, 
lassitude, muscle cramps, postural dizziness, or recent onset of dyspnea. If these 
clinical signs and symptoms are present, a repeat hematocrit will be ordered. If 
hematocrit is < 35%, the participant will be excluded from further study.   
 
7.4 Hypotension 
Nitroglycerin is administered sublingually during the flow mediated dilation studies.    
This may cause a fall in blood pressure and symptoms of hypotension.  To avoid 
the latter, nitroglycerin will not be administered to any participant whose systolic 
blood pressure is ≤100 mmHg. 
 
7.5 Special Protections for Adolescents as Research Subjects 
Because the study includes adolescent T1DM subjects between ages 15-18, 
special consideration has been given to provisions designed to protect children as 
outlined in 45 CFR 46, Subpart D. Because adolescent participants without T1DM 
would not meet the criteria of 45 CFR 46.406, control participants will only include 
adult participants.  
7.5.1 Justification and rationale for inclusion of adolescent subjects with T1DM 
The atherosclerotic changes of macrovascular disease in T1DM (e.g. 
increasing carotid intima media thickness) appear to begin in childhood and 
adolescence(80, 81) and morbidity and mortality risk is staggeringly high in 
younger adults.(10) 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 and an astounding 41.7 (95% CI 28.9–58.2) in females. Thus, it is 
clear that pathophysiologic mechanisms that contribute to macrovascular 

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disease are operative early in the natural history of T1DM. These same 
early pathophysiologic mechanisms have not been identified, however.  
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,(9) suggesting that exposure to 
insulin resistance begins early in the natural history of T1DM.  
To determine the root cause of T1DM insulin resistance and translate this 
information into therapies that would ameliorate insulin resistance in T1DM 
and its contribution to macrovascular disease, the endocrine and molecular 
mechanisms causing insulin resistance must be assessed early in the 
disease duration. This is because: 
1) T1DM insulin resistance appears to begin early in the disease 
process.(9, 28)  
2) The incidence of T1DM onset is highest between ages 10-14.(82) 
3) At younger ages and shorter disease durations there are minimal 
confounding covariables that would contribute to insulin resistance at a 
molecular level. These confounding covariables include co-morbidities 
associated with T1DM such as diabetic nephropathy,(83) prolonged 
exposure to hypertriglyceridemia,(84, 85) excessive BMI(86, 87) and 
visceral adiposity,(88, 89),  deleterious effects of aging and progressive 
inflammation on extracellular matrix remodeling,(90, 91) and increasing 
age per se.(92, 93)  
By studying adolescent and young adults early in the disease duration, this 
investigation will more precisely determine iatrogenic hyperinsulinemia’s 
association with insulin resistance and endothelial dysfunction at a very 
early point in the course of T1DM when confounding covariables are 
minimal. This knowledge is critical towards guiding subsequent therapy to 
mitigate insulin resistance and endothelial dysfunction, which will 
considerably reduce early mortality risk from macrovascular disease in the 
same subjects tested in the research.  
 
7.5.2 Assessment of risk to adolescent participants relative to 45 CFR part 46, 
subpart D 
 
It is our impression that T1DM subjects would fall under 45 CFR 46.406, 
as outlined in Vanderbilt’s Human Research Protections Program Policy 
Number IX.A, section II.C.  The proposed research is likely to yield 
generalizable knowledge about these adolescents’ condition. Specifically, 
 
1. The risk represents a minor increase over minimal risk. 

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­ The hyperinsulinemic, euglycemic clamp has been used safely in 
adolescents by the PI and elsewhere.  The technique has been 
performed over one thousand times in healthy adolescent subjects 
at other institutions such as the University of Minnesota,(94-97) 
hundreds of times in overweight adolescent(98-100) and normal 
weight prepubertal subjects(101, 102) at the University of 
Pittsburgh, and a similar number of times in normal weight children 
and adolescents at the National Institutes of Health.(103, 104) 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 7.1, extensive efforts have been employed to 
minimize risk of hypoglycemia and hypokalemia associated with 
hyperinsulinemia. 
­ The PI has personally performed the hyperinsulinemic, euglycemic 
clamp over 80 times in human subjects and nearly as many 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.(95) Every effort will be made 
to prevent 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. 
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. 
7.6 Discussion of Risks as Part of the Consent Process  
The study team will discuss all procedures, risks, and benefits with potential study 
participants, as part of the consent process. An IRB approved written informed 
consent document will be required for participation in this study. It is understood 
that consent is a process and not a discrete event.  A participant’s decision to 
withdraw consent 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 

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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 
8.1 Adverse Events 
 
8.2 Serious Adverse Events 
8.2.1 Defining Serious Adverse Events 
Consistent with FDA guidelines, serious adverse events (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. bronchospasms requiring 
emergency department referral, seizures that might not result in 
hospitalization). 
 
8.2.2 Assessing relationship between a SAE and relationship to study 
procedures 
An SAE's relationship to the study procedures will be assessed and graded 
as either: not related, unlikely, possible, probable, or definite. 
8.2.3 Assessing whether an AE is an anticipated problem 
Any AE will be assessed for whether or not it was an anticipated problem. 
In accordance with Department of Health and Human Services 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  

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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. 
8.2.4 Unanticipated, non-serious AEs 
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) and the IRB of any changes needed to the protocol. If needed, 
appropriate changes will be made to the consent form.   
8.2.5 Unanticipated SAEs 
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 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.   
8.2.6 Adverse Event Reporting 
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 whowill fund the PI on a K23 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 (or designated MD, NP, or PA KSP) medical judgement is that 
participation places the subject at risk for harm 

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10.0 
Statistical Considerations 
10.1 STUDY 1-7 (Aims 1-2) 
10.1.1 Sample Size Justification 
My accrual goal is to obtain an adequate number of participants within both 
the T1DM SUBSTUDY (STUDY 1-3) and CONTROL SUBSTUDY (STUDY 
4-7) to ensure greater than 80% statistical power (1-β) for a two-sided α-
level of 5% for each specific aim’s primary outcome. The sample size 
calculations used the best-available previously published descriptions of 
insulin sensitivity(28, 105) and %FMD(7, 30, 106-108) in T1DM. Table 7 
summaries these calculations. 
 

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Obrien-Fleming bounds for each aim’s primary analysis with a two-sided 
test with α-level of 5%, a critical z-value of 2.80 (corresponding to a p-value 
of 0.0051) will be used to end the study at interim analysis.  We will analyze 
our study’s variance and adjust the sample size if needed. 
 
10.2 STUDY B-C (Aim 3) 
10.2.1 Sample size justification 
My accrual goal for STUDY B-C is to have a sufficient sample size to 
conduct multivariable linear regression with 4 degrees of freedom to 
quantify the effect of the independent variable, mean TDDinsulin, on the 
dependent variable, GIR during the last 30 minutes of the clamp study. At 
a minimum, 10 participants are needed per degree of freedom to conduct 
this analysis, thus, we will study 30 participants with T1DM and 10 control 
participants who are otherwise healthy.  
Based on Del Prato et al.,(27) I anticipate participants completing the SCD 
arm will not see a change in GIR during the last 30 minutes of the clamp 
study compared to baseline, but will see an increase in GIR of at least 0.6 
mg/kg/min after completing the LCD arm. This study also suggests the 
pooled standard deviation of GIR will be 0.8 mg/kg/min. If the true 
difference in the ΔGIR between LCD and SCD arms for the 30 T1DM 
participants is 0.6 mg/kg/min, we will be able to reject the null hypothesis 
that the difference is zero with probability (power) 0.976. The type 1 error 
probability associated with this test of this null hypothesis is 0.05.  
10.2.2 Analysis Plan 
The correlation between the independent variable TDDinsulin (averaged over 
7 days prior to each STUDY B-C visit) and the GIR over the last 30 minutes 
of the clamp will be determined. Several other explanatory variables could 
confound this bivariate analysis, however. For example, confounding 
variables might include treatment order, hyperglycemia, hyperlipidemia, or 
measures of glycemic variability. For this reason, as with STUDY 1-5, I will 
use multivariable linear regression analysis to account for the effect of 
these potential confounders.  
10.2.3 Recruitment goals 
Based on Yki-Jarvinen et al., I anticipate a 15% drop out rate.(28) Thus, 35 
T1DM and 12 control participants will be enrolled to achieve the recruitment 
goal of 30 T1DM and 10 control participants completing the study.  
10.2.4 Interim assessment 
I will make one interim assessment after reaching one-half of the target 
accrual for the T1DM and control participants. Using Obrien-Fleming 
bounds for each aim’s primary analysis with a two-sided test with α-level of 

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5%, a critical z-value of 2.80 (corresponding to a p-value of 0.0051) will be 
used to end the study at interim analysis.  We will analyze our study’s 
variance and adjust the sample size if needed. 
 
 
 
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 up to four years.  The study results will be maintained 
indefinitely for research purposes. 

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Protocol Date:  July 29, 2021 
 
APPENDIX 1: Overnight Insulin Infusion Prior to 
STUDY 1-3 visits (T1DM Participants only) 

Protocol Version #:6 
 
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Protocol Date:  July 29, 2021 
 
Overnight Insulin Infusion Protocol 
 
The following insulin infusion protocol is intended for use in T1DM participants in the setting of the 
overnight insulin infusion prior to hyperinsulinemic clamp studies.  Target blood glucose is 90-119 mg/dL.  
 
Initiating the Insulin Infusion 
1. START TIME: the overnight infusion should begin once IV access is obtained the evening before 
the clamp study unless directed otherwise.  
2. TWO PHASES OF INSULIN INFUSION: The IV insulin infusion will proceed in two phases. 
First, we will infuse insulin at a constant rate from the time IV access is obtained after admission 
through midnight. This insulin infusion rate will equal the basal infusion rate provided by the 
participants’ insulin pumps at home. Second, beginning at midnight and continuing through the 
clamp study in the morning, we will adjust the insulin infusion rate each hour as needed to reach a 
constant blood glucose concentration of 90-119 mg/dL. 
3. PREPARATION FOR INFUSION: insulin will be dispensed as 100 units diluted in 100 mL of 
0.9% NaCl.   
a. 
PRIMING: flush 10 mL of insulin infusion through all IV tubing before insulin infusion 
begins (to saturate insulin binding sites in the tubing). 
b. CARRIER FLUID: 0.9% NaCl fluid at 20 mL/hr can be run with the IV insulin infusion 
to keep the vein open. 
c. 
HYPOGLYCEMIA PREVENTION: 20% dextrose solution should be hung and ready in 
case of hypoglycemia. Glucagon should be within easy reach. 
4. TRANSITIONING FROM HOME INSULIN REGIMEN: T1DM participants will use an insulin 
pump at home.When IV access is obtained and you are ready to begin the IV insulin infusion, ask 
the participant to suspend and disconnect his or her insulin pump immediately before starting the 
overnight insulin infusion 
5. INITIAL IV INSULIN INFUSION RATE: initial infusion rate will equal the basal rate being 
infused by the participant at home using their insulin pump.  
Example:  
Home insulin pump basal rate = Humalog 0.8 units/hr 
Initial IV insulin infusion rate = 0.8 unit/hr 
Blood glucose (BG) Monitoring 
1. Check BG hourly on the hour beginning at 2300 and record BG and insulin infusion rate on the 
task flowsheet on the designated line. 

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Protocol Date:  July 29, 2021 
 
 

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Protocol Date:  July 29, 2021 
 
APPENDIX 2: EXAMPLE OF DE-IDENTIFIED .CSV FILE EXPORTED FROM 
MYFITNESSPAL 
 
Date
Meal
Time
Calories
Fat (g)
Saturated Polyunsat MonounsaTrans Fat Cholester Sodium (mPotassiumCarbohydrFiber
Sugar
Protein (g Vitamin A Vitamin C Calcium
Iron
Note
10/30/2019 Breakfast
7:45 AM
500
28
10
0
0
0
250
1120
0
47
2
15
18
6
0
10
15
10/30/2019 Lunch
1:00 PM
636
27.8
9
0
0
0
62
1161.2
640
70.8
7.6
1.1
28.2
120
90
8
14
10/30/2019 Dinner
8:00 PM
1360
53.5
19.3
0
0
0
135
2860
0
154.5
19
15
65
75
61
40
55
10/30/2019 Snacks
3:50 PM
250
13
5
0
0
0
5
25
0
30
2
25
5
0
0
4
2
10/30/2019 Snacks
4:00 PM
480
25
4
2.5
3
0
0
600
370
51
7
26
19
10
10
22
18
10/30/2019 Snacks
10:30 PM
175
8
1.3
0
0
0
0
57.5
160
25
5
17.5
3.5
2
4
0
2
10/31/2019 Breakfast
7:35 AM
450
20
8
0
0
0
30
1370
0
48
2
6
16
2
0
6
25
10/31/2019 Lunch
636
27.8
9
0
0
0
62
1161.2
640
70.8
7.6
1.1
28.2
120
90
8
14
10/31/2019 Dinner
7:05 PM
1060
61
21.5
0
0
0
135
2325
0
79
5
8
48
142
26
70
22
10/31/2019 Snacks
3:00 PM
450
22
11
0
0
0
124
525
450
54
3
37
13
10
4
36
10
11/1/2019 Breakfast
9:35 AM
900
72
25.5
0
0
0
865
1910
0
11
0
4
50
0
0
0
0
11/2/2019 Breakfast
9:05 AM
716.7
65.5
20.8
9
18
2
907.5
1226.7
625
7.7
0.7
5.5
46.2
35
0
20
23
11/2/2019 Lunch
11:45 AM
1195
64
23
0
0
1
177
1162
0
93
4
0
57
0
20
2
4
11/3/2019 Breakfast
438.9
23.1
7
0
14
0
65
1571.6
124
81.4
4.1
15.7
9.4
6.2
31
8.8
4.1
11/3/2019 Breakfast
7:55 AM
210
21
6
4.5
8.1
1.2
507
264
243
0
0
2.4
18
21
0
12
12
11/3/2019 Snacks
9:55 AM
190
2
0
0
0
0
5
110
0
37
1
31
11
10
0
35
15
11/4/2019 Breakfast
440
20
8
0
0
0
30
1370
0
48
2
6
16
2
0
6
25
11/4/2019 Lunch
12:00 PM
510
18
7.5
0
0
0
90
1380
0
54
3
3
36
0
0
0
0
11/4/2019 Dinner
7:30 PM
740
27
15
2
8
0
190
1218
508
64
4
4
61
16
16
12
22
11/5/2019 Breakfast
7:20 AM
545
33
14
0
0
0
250
1170
0
45
2
11
18
6
2
10
20
11/5/2019 Lunch
12:40 PM
636
27.8
9
0
0
0
62
1161.2
640
70.8
7.6
1.1
28.2
120
90
8
14
11/5/2019 Dinner
5:25 PM
1170
60
21
0
0
1
255
1770
0
112
8
13
43
0
20
2
4
11/6/2019 Breakfast
7:50 AM
500
28
10
0
0
0
250
1120
0
47
2
15
18
6
0
10
15
11/6/2019 Lunch
12:30 PM
510
18
7.5
0
0
0
90
1380
0
54
3
3
36
0
0
0
0

Protocol Version #:6 
 
56 
Protocol Date:  July 29, 2021 
 
CITATIONS 
 
1. 
Cefalu WT, Tamborlane WV, Skyler JS. Type 1 diabetes at a crossroads! Diabetes 
Care. 2015;38(6):968-70. doi: 10.2337/dc15-0615. PubMed PMID: 25998288. 
2. 
National Diabetes Statistics Report, 2014: Center for Disease Control;  [April 14, 
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Livingstone SJ, Levin D, Looker HC, Lindsay RS, Wild SH, Joss N, Leese G, Leslie 
P, McCrimmon RJ, Metcalfe W, McKnight JA, Morris AD, Pearson DW, Petrie JR, Philip 
S, Sattar NA, Traynor JP, Colhoun HM, Scottish Diabetes Research Network 
epidemiology g, Scottish Renal R. Estimated life expectancy in a Scottish cohort with type 
1 diabetes, 2008-2010. JAMA. 2015;313(1):37-44. doi: 10.1001/jama.2014.16425. 
PubMed PMID: 25562264; PMCID: PMC4426486. 
4. 
Lind M, Svensson AM, Kosiborod M, Gudbjornsdottir S, Pivodic A, Wedel H, 
Dahlqvist S, Clements M, Rosengren A. Glycemic control and excess mortality in type 1 
diabetes. The New England journal of medicine. 2014;371(21):1972-82. doi: 
10.1056/NEJMoa1408214. PubMed PMID: 25409370. 
5. 
Pihoker C, Gilliam LK, Ellard S, Dabelea D, Davis C, Dolan LM, Greenbaum CJ, 
Imperatore G, Lawrence JM, Marcovina SM, Mayer-Davis E, Rodriguez BL, Steck AK, 
Williams DE, Hattersley AT, Group SfDiYS. Prevalence, characteristics and clinical 
diagnosis of maturity onset diabetes of the young due to mutations in HNF1A, HNF4A, 
and glucokinase: results from the SEARCH for Diabetes in Youth. The Journal of clinical 
endocrinology and metabolism. 2013;98(10):4055-62. doi: 10.1210/jc.2013-1279. 
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6. 
Jarvisalo MJ, Raitakari M, Toikka JO, Putto-Laurila A, Rontu R, Laine S, Lehtimaki 
T, Ronnemaa T, Viikari J, Raitakari OT. Endothelial dysfunction and increased arterial 
intima-media thickness in children with type 1 diabetes. Circulation. 2004;109(14):1750-
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15023875. 
7. 
Shivalkar B, Dhondt D, Goovaerts I, Van Gaal L, Bartunek J, Van Crombrugge P, 
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doi: 
10.1016/j.amjcard.2005.07.111. 
PubMed 
PMID: 
16377288. 
8. 
Singh TP, Groehn H, Kazmers A. Vascular function and carotid intimal-medial 
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9. 
Nadeau KJ, Regensteiner JG, Bauer TA, Brown MS, Dorosz JL, Hull A, Zeitler P, 
Draznin B, Reusch JE. Insulin resistance in adolescents with type 1 diabetes and its 
relationship to cardiovascular function. The Journal of clinical endocrinology and 
metabolism. 2010;95(2):513-21. doi: 10.1210/jc.2009-1756. PubMed PMID: 19915016; 
PMCID: 2840859. 
10. 
Laing SP, Swerdlow AJ, Slater SD, Burden AC, Morris A, Waugh NR, Gatling W, 
Bingley PJ, Patterson CC. Mortality from heart disease in a cohort of 23,000 patients with 
insulin-treated diabetes. Diabetologia. 2003;46(6):760-5. Epub 2003/05/30. doi: 
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Laing SP, Swerdlow AJ, Carpenter LM, Slater SD, Burden AC, Botha JL, Morris 
AD, Waugh NR, Gatling W, Gale EA, Patterson CC, Qiao Z, Keen H. Mortality from 
cerebrovascular disease in a cohort of 23 000 patients with insulin-treated diabetes. 

Protocol Version #:6 
 
57 
Protocol Date:  July 29, 2021 
 
Stroke; a journal of cerebral circulation. 2003;34(2):418-21. Epub 2003/02/08. PubMed 
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Protocol Version #:6 
 
61 
Protocol Date:  July 29, 2021 
 
1. 
Cefalu WT, Tamborlane WV, Skyler JS. Type 1 diabetes at a crossroads! Diabetes 
Care. 2015;38(6):968-70. doi: 10.2337/dc15-0615. PubMed PMID: 25998288. 
2. 
National Diabetes Statistics Report, 2020 Atlanta, GA: Center for Disease Control;  
[March 21, 2020]. Available from: https://www.cdc.gov/diabetes/data/statistics/statistics-
report.html. 
3. 
Livingstone SJ, Levin D, Looker HC, Lindsay RS, Wild SH, Joss N, Leese G, Leslie 
P, McCrimmon RJ, Metcalfe W, McKnight JA, Morris AD, Pearson DW, Petrie JR, Philip 
S, Sattar NA, Traynor JP, Colhoun HM, Scottish Diabetes Research Network 
epidemiology g, Scottish Renal R. Estimated life expectancy in a Scottish cohort with type 
1 diabetes, 2008-2010. JAMA. 2015;313(1):37-44. doi: 10.1001/jama.2014.16425. 
PubMed PMID: 25562264; PMCID: PMC4426486. 
4. 
Lind M, Svensson AM, Kosiborod M, Gudbjornsdottir S, Pivodic A, Wedel H, 
Dahlqvist S, Clements M, Rosengren A. Glycemic control and excess mortality in type 1 
diabetes. The New England journal of medicine. 2014;371(21):1972-82. doi: 
10.1056/NEJMoa1408214. PubMed PMID: 25409370. 
5. 
Pihoker C, Gilliam LK, Ellard S, Dabelea D, Davis C, Dolan LM, Greenbaum CJ, 
Imperatore G, Lawrence JM, Marcovina SM, Mayer-Davis E, Rodriguez BL, Steck AK, 
Williams DE, Hattersley AT, Group SfDiYS. Prevalence, characteristics and clinical 
diagnosis of maturity onset diabetes of the young due to mutations in HNF1A, HNF4A, 
and glucokinase: results from the SEARCH for Diabetes in Youth. The Journal of clinical 
endocrinology and metabolism. 2013;98(10):4055-62. doi: 10.1210/jc.2013-1279. 
PubMed PMID: 23771925; PMCID: PMC3790621. 
6. 
Jarvisalo MJ, Raitakari M, Toikka JO, Putto-Laurila A, Rontu R, Laine S, Lehtimaki 
T, Ronnemaa T, Viikari J, Raitakari OT. Endothelial dysfunction and increased arterial 
intima-media thickness in children with type 1 diabetes. Circulation. 2004;109(14):1750-
5. Epub 2004/03/17. doi: 10.1161/01.CIR.0000124725.46165.2C. PubMed PMID: 
15023875. 
7. 
Shivalkar B, Dhondt D, Goovaerts I, Van Gaal L, Bartunek J, Van Crombrugge P, 
Vrints C. Flow mediated dilatation and cardiac function in type 1 diabetes mellitus. Am J 
Cardiol. 
2006;97(1):77-82. 
doi: 
10.1016/j.amjcard.2005.07.111. 
PubMed 
PMID: 
16377288. 
8. 
Singh TP, Groehn H, Kazmers A. Vascular function and carotid intimal-medial 
thickness in children with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 
2003;41(4):661-5. PubMed PMID: 12598080. 
9. 
Nadeau KJ, Regensteiner JG, Bauer TA, Brown MS, Dorosz JL, Hull A, Zeitler P, 
Draznin B, Reusch JE. Insulin resistance in adolescents with type 1 diabetes and its 
relationship to cardiovascular function. The Journal of clinical endocrinology and 
metabolism. 2010;95(2):513-21. doi: 10.1210/jc.2009-1756. PubMed PMID: 19915016; 
PMCID: 2840859. 
10. 
Laing SP, Swerdlow AJ, Slater SD, Burden AC, Morris A, Waugh NR, Gatling W, 
Bingley PJ, Patterson CC. Mortality from heart disease in a cohort of 23,000 patients with 
insulin-treated diabetes. Diabetologia. 2003;46(6):760-5. Epub 2003/05/30. doi: 
10.1007/s00125-003-1116-6. PubMed PMID: 12774166. 
11. 
Laing SP, Swerdlow AJ, Carpenter LM, Slater SD, Burden AC, Botha JL, Morris 
AD, Waugh NR, Gatling W, Gale EA, Patterson CC, Qiao Z, Keen H. Mortality from 
cerebrovascular disease in a cohort of 23 000 patients with insulin-treated diabetes. 
Stroke; a journal of cerebral circulation. 2003;34(2):418-21. Epub 2003/02/08. PubMed 
PMID: 12574553. 

Protocol Version #:6 
 
62 
Protocol Date:  July 29, 2021 
 
12. 
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