Testing Glial Pathways to HAAF in Human Subjects Using Carbon 13 Magnetic Resonance Spectroscopy
Conditions: Diabetes Complications, Hypoglycemia
Sponsor: Pennington Biomedical Research Center

Official Title: Testing glial pathways to HAAF in human 
subjects using 13C magnetic resonance spectroscopy  
 
 
NCT02690168 
 
Document date: September 22, 2016 
 
Document type: Study Protocol and Statistical Analysis Plan 
 
Study acronym: GLIMpSE 
 

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Protocol Title: Testing glial pathways to HAAF in human subjects using 13C magnetic 
resonance spectroscopy
Study Acronym: GLIMpSE
PI Name: David McDougal, PhD
Sub-Investigator’s Name(s):
Owen Carmichael, PhD
Leanne Redman, PhD
Kishore Gadde, MD
Protocol Version Date: September 22, 2016
Objectives
CNS metabolism is maintained at the cellular level via interactions between neurons, 
glia, and the cerebral vasculature. Previous work has identified glial metabolism as a 
potential biological substrate driving hypoglycemia-associated autonomic failure 
(HAAF). Specifically, shown in an animal model, low glucose availability induces glial 
signaling in brain regions responsible for glucodetection, while others have detected 
altered glial metabolism and substrate preference in human subjects with HAAF.  The 
alterations in glial metabolism associated with HAAF are strikingly similar to those 
induced by prolonged dietary restriction in rodents. This raises the intriguing possibility 
that HAAF may be driven by glial adaptations, normally induced only by prolonged 
starvation, which are triggered in diabetic individuals by treatment-induced exposure to 
severe hypoglycemia. 
Aim 1: Using a prospective observational study design, test whether a 72 hour fast will 
cause acute alterations in human glial metabolism.  
Hypothesis: Prolonged fasting will induce changes in glial metabolism similar to 
those previously measured in individuals with HAAF.  
Aim 2: Determine if variations in fasting induced hypoglycemia and hypoleptinemia are 
correlated with changes in glial adaptation.
Hypothesis: The magnitude of the hypoglycemia and hypoleptinemia experienced 
during fasting will predict the level of glial adaptation measured in each subject. 
Background
Although diabetes is largely regarded as a disease of hyperglycemia, diabetics are 
particularly vulnerable to treatment induced hypoglycemia. Following the detection of 
severe hypoglycemia by the central nervous system (CNS), a series of physiological 
countermeasures return serum glucose to euglycemic levels. This process is rarely 
recruited in healthy adults, except in times of prolonged starvation or in endurance 
athletics (1). Yet, due to the pancreatic dysfunction associated with diabetes, diabetic 
patients are uniquely reliant on this physiological response. Tragically, repeated 
episodes of hypoglycemia leads to a progressive loss of CNS hypoglycemic counter 
regulation. This condition, termed hypoglycemia-associated autonomic failure (HAAF), 
leaves diabetic patients particularly vulnerable to increasingly severe bouts of 
treatment-induced hypoglycemia (1-4). These hypoglycemic crises are a significant 
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impediment to the maintenance of healthy plasma glucose levels in both type 1 and 
type 2 diabetics (2, 3, 5, 6), and are associated with a 3.4 fold increase risk of death (7).  
One insidious aspect of HAAF is the development of hypoglycemic unawareness, a 
situation in which the normal cues of hypoglycemia such as sweating, tremor, and 
anxiety are lost, thereby impeding a diabetic’s ability to behaviorally counteract 
hypoglycemic episodes (2, 5, 6). Results from long-term longitudinal studies suggest 
that between 6-10% of individuals with type 1 diabetes (T1DM) die as a result of acute 
hypoglycemia (8-10), undoubtedly a large majority of these deaths are caused by the 
development of HAAF (11). Although HAAF can often be reversed by prolonged 
avoidance of hypoglycemia, intensive glycemic therapies aimed at minimizing 
hyperglycemia are directly linked to increases in hypoglycemic episodes (12-15). This 
subjects individuals with diabetes to an untenable Faustian bargain, where reductions in 
long-term complications are acquired at the price of increased risk of acute death (16). 
The above only underscores the need for a more complete understanding of the 
underlying dysfunction which leads to HAAF, with the ultimate goal of reducing the 
acute risk of hypoglycemia associated with interventions utilizing tight glycemic control.
 
Although the precise mechanisms underlying the development of HAAF are unknown, it 
is thought to be driven by CNS adaptations, either in the brain cells which detect low 
glucose availability directly, or in brain regions which contain or regulate these cells (1, 
2, 4). Various mechanisms of adaptation have been proposed, which include altered 
CNS glucose utilization, modified CNS glycogen storage, and alterations in 
neurotransmission and/or neuromodulation, yet the data supporting each of these 
mechanisms are largely inconsistent (1, 4).  Recent advances in neuroimaging 
techniques have provided novel insights regarding functional and metabolic changes in 
the CNS associated with exposure to acute hypoglycemia (17). Most notably, studies 
utilizing innovative 13C magnetic resonance spectroscopy (MRS) techniques have  
demonstrated that glial metabolism is altered in humans and animals with type 1 
diabetes (18, 19), in diabetic patients with HAAF (20), as well as in animals models of 
HAAF (21). These findings suggest that the development of HAAF could be directly 
attributable to alterations in glial metabolism following exposure to hypoglycemia.  
Therefore, in order to gain a better understanding of the mechanisms underlying the 
development of HAAF, it is critical to directly link acute reductions in glucose availability 
to changes in glial metabolism in humans. This is precisely the scientific goal of this 
research proposal.
Inclusion and Exclusion Criteria
Participants will complete an initial screening (phone or online) and a screening visit at 
the Pennington Biomedical Outpatient Clinic to assess eligibility. 
Inclusion Criteria:

Male

BMI 20.0-27.9 kg/m2

18-40 years old

Willing to reside at Pennington Biomedical for 4 days
Exclusion Criteria:

Type 1 diabetes mellitus
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
Type 2 diabetes mellitus

Fasting glucose ≥ 110 mg/dL (determined at screening visit)

Hyperketonuria >15 mg/dL, (determined at screening visit)

Contraindication to MRI

History of or current eating disorder 

History of obsessive compulsive disorder

Unwilling to discontinue use  of any medication (including OTC pain medication) 
during impatient visit. 

Contraindication to prolonged fasting

Consume >10 alcoholic drinks/week

Based on the investigative team’s clinical judgement, a subject may not be 
appropriate for participation in the study.
Our eligibility criteria for the study is limited to males in an effort to reduce variability 
within our small sample size due to the known gender differences in the physiological 
response to hypoglycemia both acutely (22, 23), as well as during a 72-hr fast (24).  
Previous studies have demonstrated a reduced release of counterregulatory hormones 
(e.g. epinephrine and glucagon) in response to hypoglycemia in women. Given that this 
is a pilot study with a limited number of participants to be studied, we have chosen to 
minimize the potential variability in our outcome measures caused by these gender 
differences, by limiting our enrollment to males. This will allow us to minimize our 
participant numbers.
Number of Subjects
Up to ten subjects will be enrolled in this study.
Recruitment Methods
Standard recruitment methods (email, print, social media advertisements, etc.) will be 
used to identify potential participants. As this is a study requiring consecutive overnight 
stays at the Inpatient Unit at Pennington Biomedical, the PI may submit a privacy board 
request identifying potentially eligible participants who have previously completed 
extended-stay studies at Pennington Biomedical and have consented to future contact. 
Study Timelines
A participant’s duration of study participation will be approximately 4 days. 
The estimated duration to enroll all study subjects is anticipated to be nine months.
The estimated duration to complete the study (primary analyses) is 1 year (April 2016)
Study Endpoints
Primary endpoints: 

Percent 13C enrichment of cerebral glutamine, glutamate, and bicarbonate, as 
measured by magnetic resonance spectroscopy (MRS)  

Glial acetate oxidation rate and astroglial TCA cycle rate ascertained through 
mathematical modeling of MRS data
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Secondary endpoints:

Change in glucose levels

Change in leptin levels
Procedures Involved
The study design and schedule of assessments are depicted in Figure 1 and Table 1.
Table 1: Schedule of Assessments
Observational Study Visit 
Measurement
Screening 
Visit
Day  
0
Day  
1
Day  
2
Day  
3
Height
X
Weight
X
X
X
X
X
Medical History
X
Physical exam
X
X
Blood collection
X
X
X
X
Blood collection for future use**
X
X
X
X
X
Urine collection
X
X
X
X
X
Blood glucose*
X
X
ECG
X
CGM
X
X
X
X
Vital signs
X
X
X
X
X
13C - MRS†
X
X
DXA
X
Adverse events
X
X
X
*via finger-stick.
**Optional 
 †IV procedure and serial blood collection
CGM: Continuous glucose monitoring
Participants will complete a screening visit, and a 4-day observational inpatient stay. 
Screening: 
Participants will complete a screening visit at the Pennington Biomedical Outpatient 
Clinic to assess eligibility.  After providing written informed consent, the following 
procedures will be completed: anthropometrics, vital signs, 10ml blood draw for future 
use (optional), fasting glucose by finger stick, urine collection to measure ketonuria, 
ECG, and medical history and physical examination by a physician or nurse practitioner. 
4-Day Observational Inpatient Stay: 
Eligible subjects will be scheduled for the observational study visit (OSV) which lasts for 
approximately 80 continuous hours. The OSV begins (Day -1) with provision of a 
standard lunch and supper (Standard American Diet) to be consumed the day prior to 
the fast. Subjects will be instructed to eat only the food provided for lunch and supper, 
and arrive at the inpatient unit the following morning after at least a 10 hour overnight 
fast.  
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On Day 0, subjects will be weighed, vital signs and body composition (DXA) will be 
measured, and blood (4mL) and urine will be collected for baseline measurements of 
serum hormones, glucose, and ketones, as well as ketonuria. Blood (10 mL) will also be 
collected for future use if the participant provides permission. Baseline glial metabolism 
will be measured via MRS utilizing a simultaneous intravenous infusion of 13C labeled 
acetate. 13C-acetate will be administered for the first hour of the MRS procedure, at a 
rate of 6 mg/ kg per min for the first 5 minutes, and 3 mg/kg per min for the remainder. 
Following the MRS scan, subjects will be provided a standard breakfast, and then will 
begin ~3 days and 3 hours of fasting. During the fast, subjects will be allowed to drink 
water ad libitum and to perform normal ambulatory activity, excluding exercise.  A 
subcutaneous continuous glucose monitoring system will be utilized to track glycemia 
during the first 72 hours of the fast. After approximately 24, 48, and 72 hours of the 
initiation of the fast, blood (10mLfor study use and 10mL for future use if the participant 
provides permission) and urine will be collected, weight and vital signs measured, and 
adverse events recorded.  At 72 hours of the fast, measurement of glial metabolism will 
repeated via MRS. Subjects will then be provided breakfast (Standard American Diet, 
overfed by 50%) and discharged from the Inpatient Unit after an end of study evaluation 
(physical, blood glucose) by the Medical Investigator.
Figure 1 - Overall study design including screening and observational study visit, as well as procedures and meals. 
Abbreviations: STD (standard), WGT (weight), MRS (magnetic resonance spectroscopy scan), AEs (adverse events), 
SPC (special), EOS (end of study)
Study Procedure Descriptions

Acetate infusion/MRS procedure: Glial metabolism will be measured via MRS 
utilizing a simultaneous intravenous infusion of 13C labeled acetate. An intravenous 
catheter will be placed in a vein of each arm, one to infuse 13C-acetate and the 
other to draw blood samples. 
o
Participant alignment and baseline scans: 30 to 40 minutes prior to the start 
of acetate infusion, participants will be placed in the MRI scanner to conduct 
both preliminary alignment via MRI as well as baseline MRS scans. 
o
Acetate infusion protocol: The infusate, 350 mM [1-13C] acetate (99% 
enriched; Microbiological/Pyrogen tested), will be prepared by the PBRC 
Pharmacy. 13C-acetate will be administered for the first hour  of the MRS 
procedure, at a rate of 6 mg/kg per minute for the first 5 minutes, and 3 mg/kg 
per minute for the remainder.
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o
Blood sampling protocol:  Venous blood samples (approximately 2 mL each) 
will be obtained about every 10 minutes, starting 20 minutes before the 
beginning of the acetate infusion, and will be continued throughout the 
infusion (~15 total draws).  
o
MRS protocol: During IV infusion of [1-13C] acetate, MRS spectra will be 
collected every 10 minutes for 120 minutes using a 3 Tesla GE SIGNA MRI 
scanner housed in the PBRC Biomedical Imaging Center. 
o
Analysis: The blood collection will be used to measure changes in plasma 
acetate concentrations and % C13 enrichment of plasma acetate over time, 
via mass spectrometry.  The MRS spectra will be used to calculate changes 
in cerebral 13C enrichment of glutamine, glutamate, and bicarbonate over 
time, which reflects glial metabolism and glial amino acid synthesis.  

Anthropometrics: Fasting body weight will be collected with participants wearing a 
hospital gown and underwear. Height will be collected once at screening. 

Blood collection (study use): Approximately 37.5 mL of whole blood will be drawn 
during each acetate infusion/MRS assessment on Days 0 and 3 (described above), 
and approximately  10 mL of additional whole blood will be collected on Days 0, 1, 2, 
and 3. The total volume of blood that will be collected during the study is 
approximately  115 mL (37.5 mL + 10 mL + 10 mL + 10 mL + 10 mL + 37.5 mL).  
Blood will be collected during the MRS assessment to measure plasma acetate 
concentrations and % C13 enrichment of plasma acetate via mass spectrometry.  
Blood will be collected on Days 0, 1, 2, and 3 of the observational study to measure 
metabolic hormones of interest, such as β-hydroxybutyrate, free fatty acids, insulin, 
leptin, glucagon, glucose, and catecholamine concentrations.     

Blood collection (future use): For participants who provide permission, whole blood 
will be collected for future research (10mL at screening and  10mL at Days 0, 1, 2, 
and 3; 50ml total). Samples collected will be stored for future use by PBRC 
researchers and collaborators.

Continuous glucose monitoring: Blood glucose will be assessed using continuous 
glucose monitoring (CGM).  Briefly, the abdominal area will be disinfected, and then 
trained staff from the Inpatient Unit will insert a glucose sensor under the skin in the 
abdominal area.  The sensor has a small needle-like probe that inserts into the 
subcutaneous fat of the abdomen and that measures blood glucose levels without 
removing blood from the body.  The sensor will then be attached to the recording 
unit, and the set-up will be secured with adhesive to the participant’s body.  After an 
initial period of equilibration with interstitial glucose, the sensor will be calibrated 
about every 6-12 hours by pricking the participant’s finger to measure capillary blood 
glucose.  The CGM device records blood sugar levels every 5 minutes.

DXA: Dual X-ray absorptiometry (DXA) scans will be performed using a General 
Electric Lunar iDXA whole-body scanner (General Electric, Milwaukee, WI) during 
the first day of the Inpatient stay.  The protocol requires that subjects lie on a table 
wearing a hospital gown and no metal containing objects, while the scanner emits 
low energy X-rays and a detector passes along the body.  The scan takes 
approximately 10 to 15minutes and the radiation dose is less than 1 mrem, equal to 
about 12 hours of background radiation.  In our hands at PBRC, the standard 
deviations for DXA measurements are 320g (CVs of 0.6%) and 300g (CVs of 1.1%) 
for FFM and FM, respectively.
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
Ketonuria: Participants will collect a small urine sample in a provided collection cup 
and ketonuria will be assessed using a Ketostix® (mg/dL). 

Vital Signs: Vital signs will be collected according to PBRC standard operating 
procedures. Seated vital signs (blood pressure and heart rate) will be measured 
after a 5 minute rest.
Data and Specimen Banking
Biospecimens banked for future use will be stored indefinitely at Pennington Biomedical 
in the Clinical Chemistry and McDougal Laboratories. Data will be stored with the 
participant’s de-identified ID number. Specimens will be labeled with the participant’s 
de-identified ID number and date of collection. Data and specimens are accessible by 
the PI, his staff and designated Clinical Chemistry staff in buildings with restricted 
access. Requests for release of data and/or specimens will be considered by the PI or a 
study investigator. Upon agreement to release data and/or specimens, material and/or 
data transfer agreements will be drafted and approved by respective parties prior to the 
release of any material. 
Power analysis
The primary outcomes of this study are 13C enrichment of 
brain glutamine and glutamate, measured by MRS.  An a 
priori power analysis was performed by the Biostatistics 
and Epidemiology Core using means, standard deviations, 
and effect sizes previously reported in a similar study (20). 
This analysis estimated that a sample size of 6 would 
provide adequate statistical power for our primary outcome variables (Table 2). This 
protocol allows for enrollment of ten subjects to account for withdrawals and allow for 
complete data on six subjects. 
Data and Specimen Management
Study data collected and entered into the Pennington Biomedical Database is handled 
only by individuals with appropriate HIPAA compliance and Good Clinical Practice 
training. Participant charts and hard copy data are stored in locked offices with 
restricted access. Electronic data has exclusive restricted access granted by the 
Research Computing Group and/or the PI. For quality control, data and charts will be 
audited for completeness and accuracy (when possible). 
Study data and specimens will be stored indefinitely at Pennington Biomedical in the 
Clinical Chemistry and McDougal Laboratories. Data will be stored with the participant’s 
de-identified ID number. Specimens will be labeled with the participant’s de-identified ID 
number and date of collection. Data and specimens are accessible by the PI, his staff 
and designated Clinical Chemistry staff in buildings with restricted access.
Data analyses will be conducted as follows:
Acetate infusion/MRS procedure: Using the time courses of 13C enrichment of 
glutamate, glutamine and bicarbonate (from MRS spectra), as well as the time courses 
of plasma acetate 13C enrichments (from mass spectroscopy on plasma) we will 
Table 2. Power analysis for 
% Enrichment of Glutamine C4
Mean Diff SD
Power
N
4.0
2.3
0.845
8
4.5
2.3
0.860
7
5.0
2.3
0.849
6
5.5
2.3
0.804
5
6.0
2.3
0.863
5
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mathematically model several parameters of cerebral metabolism, such as brain acetate 
concentrations ([acetate]BR),  Glial acetate oxidation rate (Vgac), and astroglial TCA cycle 
rate (VTCAA).  Each subject will participate in two MRS scans, one immediately prior to, 
and one immediately following a 72-h fast. Repeated measures ANOVA will be used to 
compare the mean values of percent enrichment of cerebral glutamine, glutamate, and 
bicarbonate over time, as well as [acetate]BR, Vgac, and VTCAA measured before and 
after the 72-h fast, i.e., differences between first and second MRS assessments.  P-
values < 0.05 will be considered significance.  
Serum hormone and metabolite measurements:  Mean values of serum hormone and 
metabolites (including glucose) measured at each time point will be compared via 
repeated measures ANOVA.  P-values < 0.05 will be considered significance.  
Additional analysis: Correlation analysis will also be used to determine if changes in the 
parameters measured via our acetate infusion/MRS procedure are related to changes in 
our serum hormone and metabolite measurements.  
Provisions to Monitor the Data to Ensure the Safety of Subjects
We will use the definitions of Adverse Events, Serious Adverse Events, and 
Unanticipated Problems Involving Risks to Subjects or Others below. Events will be 
recorded from the participant during their inpatient stay by experienced staff trained in 
the ascertainment of adverse events from research participants. For each sign, 
symptom or adverse event, the following information will be recorded: 

A brief descriptor of the adverse event 

Start and stop dates 

Intensity (mild / moderate / severe) 

Whether the AE was “serious” or not (as defined below) 

Causal association with the intervention assigned (none / doubtful / possibly / 
probably / very likely) 

Outcome (resolved / resolved with sequelae / improving / still present and 
unchanged / death) 

Action taken with respect to the intervention (none / intervention temporarily 
discontinued / medical therapy required / intervention permanently discontinued / 
other). 
The Pennington Biomedical Research Center’s Human Research Protections Program’s 
definitions for adverse event, serious adverse event, and unanticipated problem 
involving risks to subjects or others (Policy 8) will be applied in this study and are as 
follows: 

An adverse event is any untoward physical or psychological occurrence in a 
human subject participating in research, including any abnormal sign (e.g., 
abnormal physical exam or laboratory finding, symptoms or disease associated 
with the research or the use of a medical investigational test article), symptom, or 
disease, temporally associated with the subject’s participation in the research. An 
adverse event does not necessarily have to have a causal relationship with the 
research, or any risk associated with the research or the research intervention, or 
the assessment.
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
A serious adverse event is defined as an adverse event that is fatal or life-
threatening, permanently disabling, requires or prolongs hospitalization or results 
in significant disability, congenital anomaly or birth defect.

An unanticipated problem involving risks to subjects or others is defined as 
any incident, experience, outcome or new information where all three elements 
exist:
o
Is unexpected;
o
Is related or possible related to participation in the research, and
o
Indicates that subjects or others are at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously 
known or recognized. 
While federal guidelines do not require the reporting of adverse events to the IRB, 
unanticipated problems involving risks to subjects or others will be reported to the IRB 
within 10 working days of ascertainment of the event per HRPP guidelines. 
Upon completion of each participant, participant’s anthropometric and adverse event 
data will be reviewed by the Medical Investigator with the Principal Investigator to 
evaluate the data collected regarding both harms and benefits to determine whether 
subjects remain safe. When incidental findings on imaging studies or out of range 
values on lab tests are obtained by study personnel, the participant will be notified and a 
copy of the report sent to his physician.  For lab tests, this pertains only to those tests 
for which results are obtained in real-time. 
Withdrawal of Subjects
Participants may be withdrawn from the research without their consent if they fail to 
comply with the prolonged fasting protocol or leave campus during their Inpatient stay. 
Participants that withdraw from the research will not be followed long-term as this 
protocol employs an acute intervention. 
Risks to Subjects
This study does not involve major risk to study participants.  To minimize the potential 
risks of the assessment methods and outcome variables, investigators will frequently 
monitor the study to assure that no volunteer suffers any adverse effects from 
participating in the research.  Risks of complications will be reduced by carefully 
selecting only healthy participants to enroll in the study.  The inclusion and exclusion 
criteria were created to ensure participants would have minimal risk for completing the 
study protocol. The Medical Investigator, Kishore Gadde, MD, will monitor the study 
closely and review all non-serious and serious adverse events.  Potential risks 
associated with the study procedures include (listed alphabetically):

Blood draw. There is the possibility of pain and bruising at the vein on the arm 
where the needle is inserted. Aseptic (sterile) technique and trained nursing staff 
minimizes these risks. Each acetate infusion/MRS assessment will collect 
approximately 30 mL of whole blood.  The maximum amount of whole blood 
collected throughout the 4 day evaluation period (including for future use) is 
about 165 mL. 
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
Blood pressure testing. Participants may experience discomfort during blood 
pressure recordings due to the pressure of the cuff inflating on their arm. This 
discomfort is only temporary and individuals performing this assessment are 
experienced in clinical research studies.

Body composition assessment: Dual energy x-ray absorptiometry (DXA) scans 
are determined to be minimal risk for study participants. The radiation exposure 
from a DXA scan with our equipment (the iDXA), which is 4 to 6 minutes in 
duration, is less than the equivalent of one day of environmental sun exposure.

Body weight: There is no risk to participants to have body weight measured.

13C-MRS: There are no known biological risks associated with magnetic 
resonance scanning.  It has been used routinely for over 20 years.  It produces 
side effects in very few situations.  Those situations include: 
o
Metal: Because the magnetic resonance machine uses a magnetic field, it 
can move any metallic objects that are inside the body.  This disruption of 
metal inside the body is extremely dangerous and may even be life 
threatening.  If the participant thinks he/she may have a cardiac stent, 
metallic implant, metallic piercings, shrapnel, or any other metallic material 
in the body, it is of utmost importance that the pariticpant alert the study 
coordinator or MR technician. If the participant has metallic materials in 
the body that cannot be removed, we will exclude the participant from this 
study.  
o
Electronics: Magnetic resonance imaging involves the use of radio 
frequency energy that can disrupt the functioning of electronic devices.  If 
the participant possesses a pacemaker or any other electronic medical 
device inside the body, the participant will be excluded.
o
Tattoos and cosmetics: Some tattoos and cosmetics contain metallic 
materials that can heat up during scanning, especially if they are located 
on the part of the body being scanned.  If the metallic material heats up 
enough, the participant may feel an uncomfortable burning sensation, and 
a skin burn may develop. In some cases, the amount of metallic material 
in the area being scanned is so excessive that the scan cannot proceed 
without risk of a burn developing.  In other cases, a cold compress placed 
over the metallic material can be used to prevent burning.
o
Confinement: During the MR scan, the participant will be lying down on a 
table inside of a metal tube.  The metal tube is a confined place.  This 
might produce a feeling of claustrophobia, which can be distressing.  A 
participant who has experienced claustrophobia in the past might become 
too distressed to complete the scan.  In this case, the scan will be halted.
o
Noise: The MRI machine creates a loud, rhythmic noise that sounds like 
grinding or churning.  This can be distressing to those who are sensitive to 
loud noises.  The participant will be provided with earplugs to reduce the 
noise.  But, if the participant finds the machine noises distressing, the MR 
technician can halt the scan.
o
Peripheral nerve stimulation: During the MR scan, the magnetic field 
around the body goes through rapid changes.  These changes are all 
within safety limits set by the Food and Drug Administration.  But, some 
people experience twitching in the nerves of their arms or legs as a result 
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of these magnetic field changes.  This twitching is generally not painful, 
and it stops at the end of the MR scan.  But the feeling of inadvertent 
muscle twitching may make individuals feel disoriented or uncomfortable.  
Any participant who experiences this and wishes to stop the scan as a 
result will be allowed to do so.

Continuous Glucose Monitoring (CGM):  Because CGM involves the placement 
of an implantable device below the skin, there is the possibility of discomfort, 
pain, and bruising at the site where the device is inserted.  There is also a small 
risk of bleeding and a very small risk of infection at the site of the blood draw.  
Aseptic (sterile) technique and trained personnel minimize these risks.  Finally, 
the adhesive may cause redness or irritation of the skin.

Prolonged fasting (72 hours): The risks associated with prolonged fasting are 
minimal in healthy adults, and is routinely used in clinical research studies  (e.g. 
22, 23), as well as for clinical diagnoses of insulinoma (e.g. 23, 24, 25). 
Participants are likely to experience symptoms of hypoglycemia including cold 
seats, palpitations, shakes, unsteadiness, drowsiness.  The participants will be 
continually supervised by the nursing staff in the Inpatient Unit to ensure safety, 
and any adverse events will be recorded. 

Intravenous 13C Acetate infusion: There is the possibility of pain and bruising at 
the vein where the needle associated with the IV line is inserted.  Aseptic (sterile) 
technique and trained nursing staff minimizes these risks.  We have significant 
experience in preparing and administering sterile solutions intravenously.  All 
solution will be prepared by a licensed pharmacist. 13C is a naturally occurring 
stable isotope of carbon which is commonly use in clinical research studies.  
There are no reported risks associated with administration of 13C labeled 
compounds.  The amount of acetate infused relative to blood volume is very low 
and therefore plasma pH is not anticipated to be affected.  

Urine collection: There is no risk to participants in collecting their urine. 
Potential Benefits to Subjects
There is no direct benefit to participants.
Sharing of Results with Subjects
Results will not be shared with subjects. 
Setting
This study will be conducted at Pennington Biomedical Research Center in the 
Outpatient Unit, Inpatient Unit and Bioimaging facility. Measurement of 1-13C acetate 
levels and 12C acetate levels will be completed by the Biological and Small Molecule 
Mass Spectrometry Core of the Department of Chemistry at the University of 
Tennessee-Knoxville through a contractual agreement.
Resources Available
Pennington Biomedical Outpatient and Inpatient staff are highly trained individuals with 
a breadth of experience in clinical research. All have completed human subjects 
protection training and highly knowledge of local study site and culture. A physician is 
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on-call 24 hours a day and is available for consultation or evaluations if necessary. The 
study regulatory documents will be provided to all staff involved and a startup meeting 
will be held to ensure all persons assisting with the research are adequately informed 
about the protocol, the research procedures, and their duties and functions.  
Measurement of 1-13C acetate levels and 12C acetate levels will be completed by the 
Biological and Small Molecule Mass Spectrometry Core of the Department of Chemistry 
at the University of Tennessee-Knoxville through a contractual agreement.  Specimens 
will be collected, prepared, processed, labeled, stored, and shipped by the PBRC 
Clinical Chemistry Lab.
Compensation
Participants will be paid up to $900 for their participation in the study. If for some reason 
participants are unable to complete all overnight stays and test days, they will receive 
payment for the procedures they have completed.  They  will receive up to $200 for 
completing Days 0, 1 & 2 each (including the overnight stays) and an additional $300 for 
completing through Day 3.
Confidentiality
All data and specimens will be obtained solely for research purposes. These will include 
1) physical examinations, 2) medical history, 3) blood and urine samples, 4) body 
composition measures by DXA, 5) glial metabolism by MRS, and 6) adverse events 
during study participation. All data from individual subjects will be maintained for 
confidentiality and names and identities will not be disclosed in any published 
document.
Data will be stored in hard copy in participant charts, which are kept in a locked, secure 
location only accessible to individuals with human subjects protection training. 
Biospecimens will be stored within the Clinical Chemistry Laboratory and Dr. 
McDougal’s Laboratory, both located in a building at Pennington Biomedical with 
restricted access. Data and biospecimens will be stored at Pennington Biomedical 
indefinitely however only study investigators, their staff and collaborators will have 
access to the data/biospecimens. The study PI is responsible for receipt of the data and 
biospecimens. 
All specimen samples sent to the contracted Lab for analysis will be coded with unique 
identifiers.  No personal information or personal identifiers will be sent.
Provisions to Protect the Privacy Interests of Subjects
Provisions to protect privacy interests will be untaken during this study to ensure 
participants feel at ease with the research situation. Participants will be continuously 
reminded they ask any questions or discuss any concerns in private at any time. 
Examinations, interviews and study procedures will be conducted in private rooms 
whenever possible.  
Compensation for Research-Related Injury
No form of compensation for medical treatment or for other damages (i.e., lost wages, 
time lost from work, etc.) is available from the Pennington Biomedical Research Center 
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for this study.  In the event of injury or medical illness resulting from the research 
procedures, participants will be referred to a treatment facility.  Medical treatment may 
be provided at participant’s expense or at the expense of the participant’s health care 
insurer (e.g., Medicare, Medicaid, Blue Cross-Blue Shield, Dental Insurer, etc.) which 
may or may not provide coverage.  The Pennington Biomedical Research Center is a 
research facility and provides medical treatment only as part of research protocols.  
Economic Burden to Subjects
The participants enrolled in this study are not anticipated to incur any costs during their 
participation.
Consent Process
Informed consent will be obtained from each study screener and participant prior to the 
initiation of any study procedures.  The informed consent process with take place in a 
private room at Pennington Biomedical Research Center and participants will be 
allowed ample time to read and review the informed consent documents. The informed 
consent process will be ongoing as Pennington Biomedical staff with continue to 
discuss the study, its procedures and the participants’ options throughout study 
participation. 
References
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Taborsky, G.J. and T.O. Mundinger, Minireview: The Role of the Autonomic 
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Gulanski, B.I., et al., Increased Brain Transport and Metabolism of Acetate in 
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Service, F.J. and P.C. O'Brien, Increasing serum betahydroxybutyrate 
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