Conditions: Metabolic Syndrome
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 44
Healthy volunteers: no

Brief summary:
Aim 1.Test the hypothesis that activation of the endogenous renin-angiotensin-aldosterone system impairs glycemic control via effects on insulin sensitivity and insulin secretion.

Aim 2. Test the hypothesis that activation of the endogenous renin-angiotensin-aldosterone system impairs insulin secretion and insulin sensitivity via an mineralocorticoid-receptor dependent mechanism.

Detailed description:
In aim 1 subjects are randomized to cross over between an 8 day high salt and 8 day low salt diet and assessments are made.

In aim 2, subjects are randomized to a 2x2 cross over study with an 8 day low salt diet and either eplerenone 50mg or amlodipine 5mg.

Arms:
arm 1: Subjects will be provided with a low sodium diet from the Vanderbilt Clinical Research Center that will be controlled for salt content.

Participants will be given low sodium diet (50mEq/d) and Placebo tablets for 8 days and assessments will be made, washout and then cross over to a low sodium diet (50mEq/d) plus Salt tables (150mEq) for 8days and assessments will be made. arm 2: Subjects will be provided with a diet from the Vanderbilt Clinical Research Center that will be controlled for salt content.

Participants will be given low sodium diet (50mEq/d) and Salt tables (150mEq) for 8 days and assessments will be made, washout and then cross over to a low sodium diet (50mEq/d) plus Placebo tablets for 8days and assessments will be made. arm 3: Subjects on a low salt diet will receive Epleronone 50mg for 8 days and assessments will be made, then cross over to a low salt diet with Amlodipine 5mg for 8days and assessments will be made. arm 4: Subjects on a low salt diet will receive Amlodipine 5mg for 8 days and assessments will be made, then cross over to a low salt diet with Epleronone 50mg for 8days and assessments will be made.

Interventions: intervention 1: Low Salt diet plus Placebo tablet intervention 2: Low Sodium diet plus Salt tablet intervention 3: Epleronone intervention 4: Amlodipine
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: 50mg daily intervention 4: 5mg daily

Protocol document:
Prot_SAP_000.pdf: 
The Endogenous Renin-Angiotensin-
Aldosterone System and Glucose 
Metabolism 
NCT02034435 
October 4, 2016


1 
Date: 10/4/16 
The Endogenous Renin-Angiotensin-
Aldosterone System and Glucose 
Metabolism 
James M. Luther, MD, MSCI 
 
 
Vanderbilt University Medical Center 
Nashville, TN 
 
Study Protocol  
Statistical Analysis Plans 


2 
Date: 10/4/16 
1.0 
Inclusion/Exclusion Criteria 
Inclusion criteria (Aims 1, 2) 
1)
Ambulatory subjects, 18 to 70 years of age, inclusive
2)
For female subjects, the following conditions must be met:
a. postmenopausal status for at least 1 year, or
b. status-post surgical sterilization, or
c.
if of childbearing potential, utilization of adequate birth control and
willingness to undergo urine beta-hcg testing prior to drug treatment and
on every study day.
3)
Either Metabolic Syndrome as defined by the presence of > 3 of the following:
a. Systolic Blood Pressure > 130 mm Hg OR Diastolic Blood Pressure > 85
mm Hg.
b. Glucose Intolerance (Fasting Plasma Glucose ≥ 100 mg/dL)
c.
Increased triglyceride level > 150mg/dL (1.7mmol/L)
d. Decreased levels of HDL cholesterol

For males, less than 40 mg/dL

For females, less than 50 mg/dL
e. Waist circumference

For males, greater than 40 inches (102 cm)

For females, greater than 35 inches (89 cm)
OR impaired fasting glucose (fasting glucose greater than 100 but less than 150 
mg/dL) 
Exclusion criteria (All Aims) 
Subjects presenting with any of the following will not be included in the study: 
1) Previously diagnosed type 1 Diabetes
2) Type II Diabetes, as defined during a 75g oral glucose tolerance test
performed after an 8-hour fast, by ADA criteria:
a. Hemoglobin A1C ≥6.5%
b. Fasting plasma glucose ≥126mg/dl (7.0mmol/l)
c.
2-hour glucose ≥200mg/dl (11.1 mmol/l)
d. current treatment with anti-diabetic medication(s)


 
3 
Date: 10/4/16 
3) Impaired renal function [estimated glomerular filtration rate (eGFR) of 
<60ml/min] as determined by the four-variable Modification of Diet in Renal 
Disease (MDRD) equation, where serum creatinine (Scr) is expressed in 
mg/dl and age in years: 
eGFR (ml/min/1.73m2)=175 • Scr-1.154 • age-0.203 • (1.212 if black) • (0.742 if 
female) 
4) Prior allergies to medications used in the study protocol (e.g., Amlodipine,  
Eplerenone, L-arginine, potassium chloride, insulin), or to drugs within the 
same class. 
5) Screening plasma potassium >5.5 mmol/L or sodium <135 mmol/L 
6) Cardiovascular disease such as recent (<6 months) myocardial infarction, 
presence of angina pectoris, significant arrhythmia, congestive heart failure 
(LV hypertrophy acceptable), deep vein thrombosis, pulmonary embolism, 
second or third degree heart block, mitral valve stenosis, aortic stenosis or 
hypertrophic cardiomyopathy 
7) Breast-feeding 
8) Treatment with anticoagulants  
9) History of serious neurologic disease such as cerebral hemorrhage, stroke, 
seizure, or transient ischemic attack 
10)  History or presence of immunological or hematological disorders 
11)  Diagnosis of asthma requiring use of inhaled beta agonist >1 time per week 
12) Clinically significant gastrointestinal impairment that could interfere with drug 
absorption 
13) Impaired hepatic function [aspartate amino transaminase (AST) and/or 
alanine amino transaminase (ALT) >2.0 x upper limit of normal range] 
14) Hematocrit <35%  
15) Any underlying or acute disease requiring regular medication which could 
possibly pose a threat to the subject or make implementation of the protocol 
or interpretation of the study results difficult, such as arthritis treated with 
non-steroidal antiinflammatory drugs 
16) Treatment with chronic systemic glucocorticoid therapy (more than 7 
consecutive days in 1 month) 
17) Treatment with lithium salts 
18) History of alcohol or drug abuse 
19) Treatment with any investigational drug in the 1 month preceding the study 
20) Mental conditions rendering the subject unable to understand the nature, 
scope and possible consequences of the study 
21) Inability to comply with the protocol, e.g., uncooperative attitude, inability to 
return for follow-up visits, and unlikelihood of completing the study  


 
4 
Date: 10/4/16 
 
2.0 
Enrollment/Randomization 
Ninety one subjects will be consented in AIM1 (57 subjects) and AIM2 (34 subjects). 
We estimate fifty per cent of subjects will be female. The study population will reflect 
the demographics of our area: 27% of subjects will be African American, 70% 
Caucasian, 1.6% Asian 
The protocol and rationale for assignment to treatment group will be randomized 
and blinded to the investigators and subjects. Subjects will be randomly assigned to 
low/high dietary sodium order in AIM1 and to drug order in AIM2 using a 
permuted-block randomization algorithm. Dr. Yu, study biostatistician, will provide 
an allocation schedule. The Vanderbilt Investigational Pharmacy will be responsible 
for the storage, preparation, and labeling of all investigational agents, and for 
maintaining accurate drug storage and dispensing logs. The Clinical Research 
Pharmacist will devise standard operating procedures for the pharmacy to follow 
with regard to preparing, labeling, blinding, and dispensing study drug.  
After subjects have been consented and screened, the investigator or 
research nurse will fax a copy of the consent form and a prescription containing 
check boxes for the inclusion and exclusion criteria and race to the Vanderbilt 
Investigational Pharmacy. Once the pharmacy has confirmed that consent was 
obtained and the subject met entry criteria, the pharmacist will assign the subject a 
randomization number and will provide the investigator with labeled study drug. An 
extra label containing the randomization number will be attached to each bottle of 
study drug. The investigator will affix this extra label to the subject’s records. The 
Investigational Pharmacy will retain secure documentation containing the treatment 
assignment. This will be opened in the event of a clinical scenario which 
necessitates unblinding, as determined by the PI and the safety reviewers. Subjects 
randomized in Specific Aims 1 and 2 who do not complete the whole protocol for 
any reason will be replaced and their data will be analyzed separately. 
 
3.0 
Study Procedures 
 
General Protocol 
SPECIFIC AIM 1: Test the hypothesis that activation of the endogenous renin-
angiotensin-aldosterone system impairs glycemic control via effects on insulin 
sensitivity 
and 
secretion.  
Rationale. 
 
We 
previously demonstrated 
that insulin secretion is 
deceased during low salt 
intake 
in 
healthy 
individuals; 
however, 
fasting glucose was not 
affected.  We hypothesize 
that 
insulin-sensitive 
subjects can compensate 
for 
impaired 
insulin 
 
Figure 5. AIM 1 Study design. OGTT, Oral Glucose Tolerance Test; 
HG, hyperglycemic; HI-Eu, hyperinsulinemic euglycemic; LS, low 
sodium diet (50mmol/d).  NaCl tablets=150mmol/d. 


 
5 
Date: 10/4/16 
secretion, whereas insulin-resistant subjects such as those with the MetSyn are 
susceptible to adverse effects of LS diet.  Because our animal data and studies in the 
literature suggest that Ang II and aldosterone also affect insulin sensitivity, we will also 
assess glucose turnover during hyperinsulinemic Euglycemic (HI-Eu) clamps. Because 
other compensatory effects may also alter glucose tolerance (decreased gut absorption, 
incretin signaling), we will also monitor home blood glucose serially at home to increase 
sensitivity to determine the timing of this effect.  We will avoid confounding effects of 
different diets by using the same LS diet in both arms and providing either a sodium 
chloride supplement or matching placebo to increase dietary sodium intake. 
Study Protocol.  We will complete the study in 37 subjects (of the 57 consented) with 
MetSyn using the study protocol depicted in Figure 5.  Eligible individuals must meet 
WHO criteria for MetSyn without diabetes.  Detailed inclusion and exclusion criteria 
appear in Human Subjects.  Time from initial screening to study completion for each 
subject will be 2 months (plus an additional 1 month, if drug washout needed). 
Screening visit: Subjects will be recruited by the study coordinator with use of the 
Vanderbilt GCRC volunteer database via ResearchMatch.org, Vanderbilt computerized 
medical record, e-mail, and paper flyer advertisement.  After informed consent is 
obtained, a study physician will conduct a complete history and physical exam and 
obtain screening labwork (fasting electrolytes, liver enzymes, creatinine, complete blood 
count, lipid panel, urine beta-hcg [if female], and EKG).  Subjects who meet ≥3 criteria 
for MetSyn will return to the CRC for baseline lab assessment (plasma EETs, 
aldosterone) and formal glucose tolerance testing (75g OGTT) between the screening 
day and day one diet. Body composition measurement by Dual Energy X-ray 
Absorptiometry (DEXA, see Standard Techniques), DEXA scan will be done within 3-5 
days of the hyperglycemic clamps in AIMS 1 and 2. A total of 4 DEXA scans will be 
performed on each subject who completes the study (AIMS 1 and 2.) If subjects are 
taking anti-hypertensive medications, they will wash out of these medications for 3 
weeks prior to OGTT as outlined in Standard Techniques to avoid their confounding 
metabolic effects.  Subjects with a fasting glucose >126mg/dL will be excluded.  
Subjects will be started on the study protocol within 3 months of completing the 
screening visits. 
Study Periods: After completing initial screening/baseline measurements, subjects 
will be randomized to either low sodium (LS, 50mmol/day) or high sodium (HS, 
200mmol/d) arm.  To avoid differences in diet, HS intake will be achieved by addition of 
sodium chloride supplements (150mmol/d) to achieve a total intake of 200 mmol/d; 
matching placebo will be provided during the LS period.  Subjects will be provided a diet 
containing 50mmol/d sodium, 100mmol/d potassium, and 1000mg/d calcium for 7 days.  
We chose the low dietary sodium target of 50 mmol/d to minimize blood pressure 
perturbations and reduce the risk to subjects during RAAS blockade in subsequent aims. 
A high sodium diet of 200 mmol/d suppresses the endogenous RAAS99 and maintains 
the 150 mmol/d difference between diets used in our prior study. The duration of these 
diets was determined by our previous studies which demonstrate that sodium balance is 
achieved after 5 days on LS diet and ~3 days on HS diet.  The calorie content of the diet 
will be calculated for weight maintenance (with 55% of calories from carbohydrates, 25% 
from fats, 20% from proteins).  After proper instruction by the research nurse, subjects 
will monitor glucose each day two hours after lunch and dinner during each study period.  
An 8-hour urine collection will be collected overnight prior to the hyperglycemic clamps in 
AIMS 1 and 2 for isolation of urinary exosomes. Each morning of the clamp studies, 
subjects will report to the Vanderbilt CRC in the fasting state.  Indwelling catheters will 
be placed in an antecubital vein and a hand vein.  After the subject has remained supine 


 
6 
Date: 10/4/16 
for 30-45 minutes, blood pressure and heart rate will be measured three times over ~15 
minutes with a Dinamap oscillometric sphygmomanometer.  Then blood will be obtained 
through the venous catheter for measurement of glucose, insulin, PRA, and aldosterone.  
Subjects will then undergo a hyperglycemic clamp as described under Standard 
Techniques. Subjects will return on Day 8 for HI Eu clamp to assess overall insulin 
sensitivity and hepatic 
glucose production (as 
outlined 
in 
Standard 
Techniques) 
while 
continuing study diet.  A 
24-hour 
urine 
for 
measurement 
of 
electrolytes, aldosterone, 
and creatinine will be 
collected before HI-Eu 
clamps in AIMS 1 and 2 
to 
assess 
protocol 
compliance 
and 
achievement of steady-
state sodium balance. After completion of Period 1, subjects will resume their normal 
diet (wash out) for ≥2 weeks, and then cross over to the remaining dietary sodium 
condition  
 
SPECIFIC AIM 2: Test the hypothesis that endogenous aldosterone impairs insulin 
secretion and insulin sensitivity via the mineralocorticoid receptor. 
Rationale. Hyperaldosteronism is the most common endocrine cause of hypertension, 
present in 10-20% of resistant hypertension patients, usually caused by either idiopathic 
hyperaldosteronism (IHA) or aldosterone producing adrenal adenomas (APAs).105-107 
Hyperaldosteronism is also associated with diabetes, and aldosterone decreases insulin 
sensitivity. Our in vitro studies in islets demonstrate that aldosterone impairs insulin 
secretion via oxidative stress independent of the MR. We hypothesize that MR 
antagonism will improve insulin sensitivity and insulin secretory capacity.  We will test 
this hypothesis in a group of subjects using the specific MR antagonist eplerenone (AIM 
2A)  
Study Protocol.  In Aim2, we will complete the study in 20 subjects. After screening and 
characterization, subjects will be randomized to either eplerenone 50mg daily, or 
amlodipine 5mg daily in a 2x2 crossover study as shown in Figure 6.   Subjects will be 
provided LS diet (50mmol/d) and study drug concurrently for 8 days.  Subjects will report 
to the Vanderbilt CRC after an overnight fast on the 6th day for hyperglycemic clamp and 
on the 8th day for HI-Eu Clamp.  During the LS study period, we will measure seated 
blood pressure and assess for hypotensive symptoms every day when the subjects 
report to the CRC kitchen to obtain their food.  We will provide subjects with a 24-hour 
contact number for the study coordinator and/or PI.  Based on previous studies of RAAS 
inhibition during LS diet,103 we anticipate that this will be well tolerated over the short 
period of the study.  We will further monitor plasma potassium, plasma sodium, and 
creatinine on day 4 and both study days during each period as safety measures.  24-
hour urine collections will be obtained before each study day and plasma creatinine will 
be measured to assess creatinine clearance, dietary sodium balance, and protocol 
adherence.  After completion of the first study period, the subject will stop the study 
medication, wash out for ≥2 weeks, and then begin the next treatment period. 
 
 
Figure 6. AIM2 Study design. OGTT, DEXA, Oral Glucose 
Tolerance Test; HG, hyperglycemic; HI-Eu, hyperinsulinemic 
euglycemic; LS, low sodium (50mmol/d) 


 
7 
Date: 10/4/16 
STANDARD TECHNIQUES 
Data management and quality control.  We will use the web-based Vanderbilt 
REDCap system to design an electronic data collection form. This form will be pilot-
tested before use.  Data will be input into a protected web-based case report form (which 
can be readily downloaded into SAS, STATA, R, or SPSS).  The form allows for direct 
data entry by investigators across multiple institutions and is designed to minimize errors 
and erroneous values. Results from the Vanderbilt Clinical Laboratory can also be 
directly imported to REDCap, which further reduces typographical data entry errors.  
Expected ranges are pre-specified to prevent errors such as the shifting of decimal 
points.  The program includes a computerized audit trail so that the identity of individuals 
entering or changing data and, in the case of changes, both original and revised data are 
saved.  Data are backed up daily. Clinical data, including clinical laboratory, will be 
entered by the research nurse.  Research laboratory data will be entered by a research 
assistant.  A unique identification case number will be used to protect the confidentiality 
of the study participants. The case numbers and participants' names will be included in 
the protected source database but only case numbers will be included in any exported 
data files used for the statistical analysis.  Before analysis, the data manager will 
independently and blindly assess all raw data for accuracy and completeness. Dr. 
Chang Yu, biostatistician, will check for potential outliers and resolve them with the 
investigators before unblinding the data and performing statistical analyses with input 
from Dr. Luther based on the methods specified in the data analysis plan.  We will use 
the web-based case report form to monitor quality performance.  We will generate 
monthly reports to track recruiting and safety.  
Baseline measurements. Waist (horizontal umbilicus) and hip (at the largest 
horizontal span) circumferences will be measured to 0.5 cm precision in triplicate with a 
spring-loaded tape measure (Gulick II by Country Technology, Gay Mills, WI). Body 
composition will be determined using the Bod Pod Composition System (Life 
Measurements Instruments, Concord, CA).   
Assays.  Clinical laboratory assays such as potassium concentrations will be run locally 
in the CLIA-approved laboratories.  All blood samples drawn for research assays will be 
centrifuged at 0 ºC for 20 minutes immediately after blood drawing, and plasma or serum 
will be divided into two aliquots, labeled with the case identification number, and logged 
and stored separately at -80 ºC until sampling. Standard quality assurance measures will 
be applied to all laboratory analyses including, but not limited to, tracking of inter-assay 
variability and the reanalysis of blinded internal controls at different times. 
Glucose, C-peptide, and Insulin: Plasma glucose will be measured by the 
glucose oxidase method with a YSI glucose analyzer (YSI Life Sciences, Yellow Springs, 
OH).  6,6-dideuterated glucose will be assessed using mass-spectrometry.  Plasma 
insulin concentrations will be determined by radioimmunoassay (RIA; Millipore, St. 
Charles, MO).11,113  The insulin assay cross-reacts with 38% intact human pro-insulin 
and with C-peptide, ≤0.01%.  Samples for C-peptide and glucagon will be drawn into 
heparinized tubes containing 250 KIU Trasylol (Aprotinin) per mL of whole blood. This 
will result in a final concentration of approximately 500 KIU Trasylol per mL of serum or 
plasma. C-peptide will be measured using RIA (Millipore).11  Urinary catecholamines will 
be analyzed by HPLC as previously described.75   
Lipid Analysis: Total cholesterol, triglycerides, and HDL will be determined using 
automated techniques (UniCel® DxC 800 Synchron® Clinical System, Beckman Coulter). 
We will measure non-esterified free fatty acids using a commercially available enzymatic 
assay (Wako Chemicals, Dallas, TX). 
Aldosterone will be determined using a radioimmunoassay utilizing 
125I-


 
8 
Date: 10/4/16 
aldosterone (MP Biomedicals, Irvine, CA), a primary antibody to aldosterone (NIDDK 
National Hormone & Peptide Program, Torrance, CA), and a secondary anti-rabbit 
gamma globulin antibody (Linco Research, St. Charles, MO).11,75,114-116  Plasma renin 
activity (PRA) will be determined by radioimmunoassay (DiaSorin, Stillwater, MN).  
DNA samples: A DNA sample will be drawn only once, at the beginning of the study, 
and will be storage for genotypification of SNPs related to impaired β-cell function. The 
DNA samples analysis will be undertaken in a different study, given that it goes beyond 
the scope of the present study. Genotyping will be performed for research purposes 
only, and subjects will not be informed of results. 
Medication withdrawal (hypertensive subjects). Anti-hypertensive medications 
will be discontinued three weeks prior to initiation of study drug. Because we have 
found that spironolactone has a prolonged duration of effect requiring a longer washout 
period, this medicine will be discontinued 4 weeks prior to initiation of study drug. 
Whenever appropriate, medications will be tapered. During this period, blood pressure 
will be measured every 1 to 3 days. If the subject does not have a suitable home blood 
pressure monitor, we will provide one for temporary use during the study.  Home blood 
pressure monitors will be confirmed to vary ±<5mmHg for both SBP and DBP in 
comparison to the Dinamap reading.  If any seated systolic pressure is >180 mmHg or 
the seated diastolic pressure is >110 mmHg or if a hypertensive subject develops 
symptoms of high blood pressure (e.g. headache, vision disturbance, chest pain, or 
dyspnea) regardless of the pressure, that subject will be discontinued from the study 
and his or her anti-hypertensive medications will be restarted. In our experience, 
approximately 5 per cent of enrolled subjects may be excluded by this blood pressure 
criterion. Excluded subjects will be replaced. 
Blood pressure measurements. During office visits and studies, blood pressure will 
be measured with an automated oscillometric recording device (Dinamap, Critikon, 
Carlsbad, CA). At the time the cuff is placed, its readings will be compared against those 
of the mercury sphygmomanometer. In our experience these two methods give readings 
within 2-4 mm Hg of each other for both systolic and diastolic pressures. In our 
experience, this method correlates with average blood pressure obtained during 24-hour 
ambulatory blood pressure monitoring (r=0.51, P<0.001). 
Oral Glucose Tolerance Tests (OGTT). Oral glucose tolerance tests will be 
administered according to guidelines by the American Diabetes Association to determine 
the baseline metabolic characteristics of the subjects. An IV catheter will be inserted for 
measurements. Baseline glucose and insulin will be drawn after an overnight 8-hr fast, 
then 75g glucose solution will be administered orally. Blood samples will be collected 
every 30 minutes for a total of 120 minutes for glucose and insulin. Subjects will be 
classified according to Table 1.  
 
Table 1 OGTT Classification (75g) 
Time Point 
Glucose 
(mg/dL) 
Classification 
0 hours (Fasting 
Glucose) 
<100 
Normal Fasting Glucose 
100-125 
Impaired Fasting Glucose 
≥126 
Diabetes 
2 hours 
<140 
Normal Glucose Tolerance 
140-199 
Impaired Glucose Tolerance 
≥200 
Diabetes 


 
9 
Date: 10/4/16 
Home blood glucose monitoring. Subjects will be trained to properly measure home 
blood glucose by a trained research nurse, and will be provided a logbook for recording 
results, time of day, and timing of standardized meal ingestion. Subjects will be provided 
a Freestyle Freedom Lite glucose monitor (Abbott), alcohol wipes, single-use lancets, 
and test strips. Subjects will be directly observed performing the initial measurement to 
verify proper sterile technique, and printed manufacturer instructions will be provided 
for future reference. Between subjects, glucose monitors will be sterilized with alcohol 
pads and battery status will be checked and replaced near the end of life. Dedicated 
clinical glucose monitors will be kept in the Vanderbilt GCRC. 
Body Composition Measurement.  Body composition will be measured by Dual 
Energy X-ray Absorptiometry (DEXA). The computer software permits measurements of 
the whole body as well as individual segments, this is an accurate, validated method 
with minimal radiation exposure (Mazess et al., 1990;Svendsen et al., 1993). A Lunar 
model DPX Absorptiometer (Lunar DPA software 3.6; GE Medical System, Madison, WI), 
a research scanner located in the Vanderbilt GCRC will be used to measure total and 
regional adiposity.  
Resting Energy Expenditure measurement. Resting energy expenditure will be 
measured using indirect calorimetry  with a ventilated canopy device (CPX/D system, 
Medical graphics Corporation, St Paul, MN), this is based on the calculation of heat 
production through the measurements of gas exchange, specifically oxygen consumption 
and carbon dioxide production. Both measurement are converted to energy expenditure 
by application of deWeir’s formula (deWeir, 1949). This method has been validated 
previously (Isbell et al., 1991) and proved to be accurate for resting energy expenditure 
determination.  Only the last 20 minutes of a 40-minute measurement period will be 
analyzed. The respiratory quotient RQ will be obtained by dividing VCO2 by VO2.  The RQ 
will be used as an indicator of quality control because in humans it resides in a fairly 
narrow range (0.67-1.3) (Branson, 1990).   
Assessment of Insulin secretion by Hyperglycemic Clamp. After an overnight 
fast, primed continuous glucose will be infused for 2 hours via an antecubital vein, per 
the methods of DeFronzo et al, and Elahi et al. Arterialized blood will be obtained by 
inserting a venous cannula into a hand vein in a retrograde fashion and then placing the 
hand into a heated box or under a heating pad at 60ºC. If a retrograde hand vein 
catheter is not obtained, an antecubital vein may be used instead. Blood sampling will 
be done according to Figure 7, and the exogenous infusion of 20% dextrose will be 
varied to maintain the plasma glucose at a target glycemia of 200 mg/dL for 150 
minutes, using a standardized algorithm as described by DeFronzo.93 Blood samples will 
be collected every 5 minutes, immediately centrifuged, and plasma glucose will be 
determined with a YSI glucose analyzer (average time ~2 minutes to result). Samples 
for insulin will be collected (t= -20, -10, 0, 2.5, 5, 7.5, 10, 15, 20, 30, 60, 90, 105, 120, 
122.5, 125, 127.5, 130, 135, 140, and 150) and stored at -80ºC until assay. At 120 
minutes, an intravenous L-arginine hydrochloride bolus (5 g) will be administered over 5 
minutes to assess non-glucose stimulated insulin release.94 After completion of the 
study, dextrose will be tapered, and plasma glucose will be monitored for signs of 
hypoglycemia for 2-3 hours. The acute insulin response (AIR) will be defined as the 
incremental change for insulin for time 0-10 minutes following initial glucose bolus. 
Second phase insulin response (ΔInsulin120) will be calculated as the incremental change 
in plasma insulin during steady state (time period 90 to 120) compared to baseline 
values. L-Arginine stimulated insulin secretion will be calculated as the incremental 


 
10 
Date: 10/4/16 
change 0-10 minutes after injection compared to steady state (average during time 
period 90-120 min.). To ensure that changes of insulin concentration are not due to 
altered 
metabolism, 
C-peptide 
will 
be 
assessed 
simultaneously. 
 
Assessment of Insulin sensitivity by hyperinsulinemic-euglycemic clamp.  An 
intravenous catheter will be placed in the antecubital vein of the non-dominant arm, and 
a retrograde catheter will be inserted into the contralateral hand vein.  If a hand vein 
catheter cannot be maintained, a one will be inserted into a forearm or antecubital vein. 
Following the placement of catheters, subjects will be allowed to rest at least 30 minutes 
before baseline measurements are made.  Insulin will be infused in the arm contralateral 
to the sampling venous catheter. Insulin infusion rates were chosen to partially suppress 
hepatic glucose production during the low dose infusion, while completely suppressing 
hepatic glucose production and maximally stimulating peripheral glucose utilization 
during the high dose infusion in insulin resistant subjects.119-122 Resting Energy 
Expenditure (REE) measurement will be performed during the equilibration period of HI-
Eu clamp in both AIMS 1 and 2. A priming insulin dose will be given at the time of each 
insulin infusion change. Plasma glucose will be measured every 2.5 to 5 minutes and an 
exogenous infusion of 20% glucose will be adjusted to control the rate of fall of glucose 
and maintain the plasma glucose at a target glycemia of 90 mg/dL by a modification of 
the glucose clamp technique (Figure 8).123,124 Potassium chloride (KCl) 40mEq will be 
administered orally prior to initiation of glucose/insulin infusion during each 
hyperinsulinemic test period to maintain plasma potassium levels.  Serum potassium will 
be measured prior to insulin initiation,during, and at the end of the study, and KCl will be 
administered to keep K+>3.8. Heart rate and ECG will be recorded continuously during 
all glucose clamp studies.  
Glucose Turnover: During hyperinsulinemic-euglycemic clamps, endogenous 
glucose appearance (EndoRa) and utilization (Rd) will be determined by stable isotope 
 
Figure 7. Hyperglycemic clamp protocol. Plasma glucose is measured every 5 
minutes throughout the study, and dextrose infusion rate is adjusted. Blood sampling 
for insulin and C-peptide (▲). 
 
Figure 8.  Two-step hyperinsulinemic euglycemic clamp protocol.  Plasma glucose is measured every 5 
minutes throughout the study, and dextrose infusion rate is adjusted.  Blood sampling for glucose (▲). 


 
11 
Date: 10/4/16 
enrichment with 6,6-dideuterated glucose.125  In the proposed experiments, total rates of 
appearance (Ra) and Rd will be calculated based on the equations of Steele et al.126 as 
modified by Debodo.127  During hyperinsulinemic glucose clamps, Ra and Rd, calculated 
using this approach, may be underestimated.128 The error appears to be greatest in 
states of high glucose flux.129  We will use 6,6-dideuterated glucose as the glucose 
tracer to avoid calculation errors due to glucose contamination.  To minimize the effect of 
rapidly changing enrichment130, conclusions based on glucose turnover will only be 
made when steady state conditions are proven to exist. This will include the end of a 120 
min tracer equilibration period and during the last 30 min of the hyperinsulinemic-
euglycemic clamps, when a steady state exists.  We define steady state as a period 
when the coefficient of variation of the plasma glucose and enrichment are stable at 
<3%. Insulin sensitivity will be estimated by the rate of glucose infusion (M) necessary to 
maintain euglycemia during steady-state (terminal 30 minutes of the study).  To control 
for inter-individual variations in plasma insulin concentration, the insulin sensitivity index 
will be calculated by dividing M by average steady state insulin concentration.  Tracer 
delivery will be varied to ensure that plasma enrichment changes very little.  In 
experiments in which the change in glucose flux is so minimal that no exogenous 
glucose is required, the tracer will be administered as a primed continuous infusion (3.6 
mg/kg bolus followed by 0.06 mg/kg/min constant infusion.  Using this methodology, Dr. 
Ikizler has determined that endogenous glucose disposal rate is greatly diminished in 
obese chronic hemodialysis patients. The Disposition Index will be calculated as the 
product of insulin secretion during hyperglycemic clamps (ΔInsulin0-10) and insulin 
sensitivity during hyperinsulinemic euglycemic clamps (M/I-high dose).  
 
4.0 
Risks 
Inconveniences: 
• 
Not eating or drinking after 10p.m. on the night before each study  
• 
Collecting urine in a jug for 24 hours  
• 
Eating a special bland diet for 7 days 
• 
Traveling to the GCRC and picking up meals and medications 
Risks: 
• 
Frequent blood draws can lead to anemia. 
• 
Putting a catheter into a vein may cause bleeding, bruising, or infection 
(uncommon). We will use careful and sterile techniques to minimize these side 
effects. 
• 
Withholding blood pressure medications may result in elevation in blood 
pressure. We will monitor blood pressure at home every 1-3 days after stopping 
or decreasing these medications. If home blood pressure is reported to be 
>180/110 mmHg, the study nurse and physician will re-evaluate them. During 
this evaluation, if any seated systolic pressure is >180 mmHg or the seated 
diastolic pressure is >110 mmHg or if a hypertensive subject develops symptoms 
of high blood pressure (e.g., headache, vision disturbance, chest pain, or 
dyspnea) regardless of the pressure, that subject will be discontinued from the 
study and his or her anti-hypertensive medications will be restarted. 
• 
Glucose: Infusion of glucose may cause hyperglycemia. Symptoms of high blood 
sugar are hunger, thirst, frequent urination, dry mouth, and dry skin. Blood will 
be checked after the study completion until blood sugar returns to the normal 
range. IV Glucose may cause irritation of the vein, including redness and pain. 
We will give the glucose through a bigger vein to decrease this risk. 


 
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Date: 10/4/16 
• 
Insulin: Persistently elevated insulin levels may cause hypoglycemia. Symptoms 
of low blood sugar are nausea, extreme hunger, feeling nervous or jittery, cold, 
clammy, wet skin and/or excessive sweating not caused by exercise, a rapid 
heartbeat, numbness or tingling of the fingertips or lips, and trembling. Blood 
glucose will be checked after the study until it returns to the normal range. 
• 
Anti-hypertensive medications (amlodipine, eplerenone): may lower blood 
pressure excessively or alter serum potassium levels. We will monitor blood 
pressure and potassium throughout the study, and supplement with potassium 
as needed.  
• 
Anti-hypertensive medications (amlodipine, eplerenone): may produce allergic 
reactions. We will exclude subjects with allergies to these drugs and if allergies 
to these drugs develop during the course of the study, we will discontinue the 
subject. 
• 
Drugs that interrupt the renin-angiotensin system (angiotensin receptor blockers, 
Eplerenone in Aim 2) have been associated with birth defects if used during 
pregnancy. As a precaution we will exclude pregnant women or those women 
not using a reliable birth control method. We will check urine pregnancy tests 
before each study day. If a woman self-reports pregnancy to us, we will 
immediately discontinue study medication and withdraw that subject. Risk is 
further minimized by the short duration of eplerenone treatment. 
• 
L-Arginine: May cause irritation of the vein. We will give it through a bigger vein 
to decrease this risk. It may cause nausea, vomiting, headache, flushing, 
numbness, or local venous irritation in ~3% of subjects using doses 6 times the 
dose that will be used in this study (30g over 30 minutes). Post-marketing 
surveillance of 1,670 infusions has revealed the following: One patient had an 
allergic reaction which was manifested as a confluent macular rash with 
reddening and swelling of the hands and face, which subsided rapidly after the 
infusion was terminated and 50 mg of diphenhydramine were administered. One 
patient had an apparent decrease in platelet count from 150,000 to 60,000. One 
patient with a history of acrocyanosis had an exacerbation of this condition 
following infusion of RGene® 10. We have not experienced any side-effects in 
our prior studies infusing L-arginine at the dose used in this study. 
• 
Potassium Chloride: Administration of potassium chloride in excessive doses can 
cause hyperkalemia. We will monitor for signs and symptoms of high blood 
potassium include weakness, cardiac arrhythmias, and numbness. The dose 
administered in this study is not expected to cause an excessive rise in 
potassium, and is necessary to prevent or treat hypokalemia due to our 
standardized diet, glucose, and/or insulin administration. Oral potassium chloride 
may cause an unpleasant taste, stomach discomfort, nausea, stomach bleeding 
or diarrhea. Intravenous potassium may cause discomfort when infused rapidly 
or in a concentrated solution. Potassium will be diluted by co-administered 
glucose, decreasing this risk.   
• 
There may be risks that we do not know about at this time. If we should find out 
any new information, we will notify the participants. 
 
5.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 


 
13 
Date: 10/4/16 
All protocols have been or will be reviewed and approved by the Vanderbilt IRB 
before any subject is enrolled.  The Principal Investigator and Co-Investigator will closely 
oversee the protocol in conjunction with the dedicated research nurse.   
We will use structured electronic case report forms and the REDCAP Dynamic Data 
Pull (DDP) within REDCap to assess compliance, concurrent medications and medical 
conditions, and adverse events.  
Any adverse events or toxicities will be reported to the IRB as per IRB guidelines.  
Any untoward medical event will be classified as an adverse event, regardless of its 
causal relationship with the study. An adverse event will be classified as serious if it a) 
results in death, b) is life-threatening, c) requires inpatient hospitalization or prolongation 
of existing hospitalization, d) results in persistent or significant disability or incapacity, e) 
is a congenital anomaly or birth defect, or f) is an important medical event based upon 
appropriate medical judgment.  Serious adverse events will be reported to the Safety 
Monitor, IRB, and NIH within 24 hours. An initial written report will be submitted within 72 
hours.   
Non-serious, unexpected adverse events will be reported to the IRB and Safety 
Monitor at the time of annual Continuing Review  AEs will be graded as Mild (no 
limitation of usual activities), Moderate (some limitation), or Severe (inability to carry out 
usual activities) and attributed according to the relationship to the study drug and/or 
procedures as definitely, probably, possibly or unrelated to the study intervention and/or 
procedures..  Summary Reports will be submitted to the IRB at least annually and will 
contain a) The number of adverse events and an explanation of how each event was 
handled, and b) The number of complaints and how each complaint was handled, c) The 
number of subject withdrawals and an explanation of why the subject withdrew or was 
withdrawn, and d) The number of protocol violations and how each was handled. 
Dr. C. Michael Stein, MD, professor of medicine and pharmacology, Dan May Chair 
in medicine, will serve as the independent Safety Monitor for these studies. Dr. Stein is 
an experienced physician scientist with extensive experience with human subject 
research and post-marketing drug surveillance.  Dr. Stein will not be involved in the 
conduct of the studies, as a key personnel on the corresponding NIH grant, or serve in a 
reporting relationship (boss or subordinate) with Dr. Luther.  As the Safety Monitor, Dr. 
Stein will annually review subject enrollment, retention, compliance, and adverse events 
in a report prepared by Dr. Luther and the study statistician.  The Safety Monitor will 
have the authority to request necessary protocol changes. Dr. Stein will additionally 
provide an annual summary report to the IRB and NIH at the time of each annual review. 
No early stopping based on efficacy is planned. Interim data will be provided as 
requested to the Safety Monitor by Drs. Luther and Yu.  
 
6.0 
Study Withdrawal/Discontinuation 
 
Subject Withdrawal 
 
Participation in this study is entirely voluntary, and subjects may withdraw from 
the study at any point by informing the study coordinator or PI. Data that have been 
collected may be used for analysis, but no further data will be collected after subject 
withdrawal. 
 


 
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Date: 10/4/16 
Subject Discontinuation 
 
Subjects may be removed from this study without consent if: 
• 
Staying in the study would be harmful to the subject 
• 
The subject no longer meet the requirements of the study 
• 
The study is stopped 
 
7.0 
Statistical Considerations 
 
Sample Size Calculations and Statistical Analysis.  Sample size calculations are 
based on our prior results, using the hyperglycemic clamp first phase insulin response 
(ΔInsulin0-10 minutes) in normal and MetSyn subjects.  This measure was chosen 
because it is the first detectable insulin secretory defect in subjects which progress to 
diabetes,65 and because it is a highly reproducible measure. In our prior studies in 
MetSyn subjects, this measure is highly correlated within subjects over the course of 3 
months (r=0.91, P<10-6).  The average response is 44±27 µU/ml in MetSyn subjects 
during HS diet, with an average coefficient of variation of 0.71 (range 0.61-0.80) across 
multiple studies.  Based on our previous observed effect of LS diet (-40% vs HS), a 
sample size of 20 will have 80% power to detect a difference in means of 11 (i.e. effect 
size of -25%, a First condition mean, µ1, of 44±32 and a Second condition mean, µ 2, of 
33±24, correlation of 0.85), using a paired t-test with a 0.05 two-sided significance level. 
In our prior studies targeting subjects with MetSyn, ~75% of those we consented met 
inclusion criteria using our recruitment and screening methods. We estimate 15% 
dropout.   
We will increase enrollment to allow for completion of 20 subjects in Aim2 based 
on the above assumed drop-out rate and consent rate. Therefore in AIM1, we will screen 
57 subjects and enroll 40 subjects in order to complete 34.   This would provide 96% 
power to detect the above difference in Aim1. In a prior study of MetSyn subjects with 
repeated measurements over 3 months, fasting glucose values were normally distributed 
with SD of 12mg/dl (CV 12%). Therefore, the detectable change in fasting glucose in this 
study with 34 subjects is 5.9 mg/dl, with 80% power and Type I error rate of 0.05 
assuming a conservatively weak within-subject correlation 0.5 for this endpoint. 
Data analysis plan. Analyses in AIM1 will focus on the primary outcomes of insulin 
secretion, insulin sensitivity, and plasma glucose and the secondary outcomes of hepatic 
glucose production and disposition index.  The within-subject design avoids inter-
individual variation in measures of insulin secretion.  The primary measure of the 
hyperglycemic clamp will be peak insulin secretion in response to glucose and L-
arginine.  Because the secretion of insulin during the study is time-dependent and a 
steady state is not achieved, we will assess baseline, acute response (ΔInsulin from 0-
10 minutes), second phase (ΔInsulin from 90-120 minute), and L-arginine peak insulin 
concentrations, insulin sensitivity index (M/I), and disposition index using mixed effects 
models.  A comparison of C-peptide will be performed to determine whether altered 
insulin metabolism accounts for the difference in insulin concentrations.  Secondary 
analyses will be performed similarly. 
Even though we do not expect any carry-over effect based on our prior 
experience with similar studies, we will nevertheless test for a carry-over effect using the 
T-test approach proposed by Jones and Kenward.100 Mixed effect models will be used to 
analyze the data with a random subject effect and with the treatment (LS vs. HS diet) 
and the time trend (baseline, 10 min, 30 min, and 60 min into the infusion) as fixed 
effects.  The focus of this study will be the treatment effect and the time trend of the 
endpoints; however, mixed effect models also provide the flexibility of controlling for and 


 
15 
Date: 10/4/16 
evaluating covariates such as age, sex, baseline insulin sensitivity, and family history of 
T2DM.  The mixed effect models are robust in the sense that they can include subjects 
with missing data at some time points but not all time points to estimate the effects of 
interest.  Besides the above evaluation of the treatment effect and the time trend through 
the regression models, specific inferences regarding effects of interest will be made by 
reporting a point estimate along with a 95% confidence interval. Hypotheses will be 
tested at the level of α=0.05. This data analysis plan will be carried out using statistical 
software SPSS for Windows (Version 19.0, SPSS, Chicago), SAS® release 9 (Cary, 
North Carolina) and the open source statistical package R (version 2.12).101 
 
 
8.0 
Privacy/Confidentiality Issues 
 
 
A unique identification case number generated by the collection program will be 
used to protect the confidentiality of the study participants. The case numbers and 
participants' names will be entered into the secure web-based database, and only the PI 
or the study nurse will have access to PHI. The GCRC database program will 
automatically exclude any PHI during export of the data to any external data files used 
for analysis. Only the PI, co-investigators, and research team listed as Key Study 
Personnel will have access to any PHI. 
 
9.0 
Follow-up and Record Retention 
 
 
The duration of this study will be ~3 years. The study results will be kept in the 
subject’s research record for at least seven years after the study is finished. At that 
time, the research data that has not been put into the computerized medical record may 
be destroyed. Any research data that has been put into the computerized medical record 
will be kept for an unknown length of time.