Use of a Hyperinsulinemic-hypyglycemic Clamp to Study Hypoglycemia: a Method Development Study
Conditions: Diabetes
Sponsor: Pennington Biomedical Research Center

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Protocol Title: Use of a hyperinsulinemic-hypoglycemic clamp to study hypoglycemia: 
a method development study. 
 
PI Name: David McDougal 
 
Sub-Investigator’s Name(s): Daniel Hsia, Leanne Redman, and Eric Ravussin. 
 
Protocol Version Date: November 15th, 2018 
 
Funding: LA CATS/Internal 
 
Objectives 
Hypoglycemic complications are a major impediment to the maintenance of healthy 
glucose levels in persons with diabetes. We recently completed a clinical pilot and 
feasibility study (GLIMPSE), which identified a novel biomarker that appears to predict 
the susceptibility to hypoglycemia [1]. By providing an assay to predict hypoglycemic 
events and therefore diabetic complications, the development of this biomarker could 
significantly improve the treatment of persons with diabetes.  
 
To pursue such line of research requires the induction and maintenance of hypoglycemia 
in a controlled safe clinical research setting. A hyperinsulinemic-hypoglycemic clamp is 
an experimental procedure, which allows for hypoglycemia to be studied in a safe and 
controlled manner [e.g. 2, 3].  The goal of this study is to establish the hyperinsulinemic-
hypoglycemic clamp procedure at Pennington Biomedical Research Center in order to 
apply the knowledge gained to future studies which will determine the efficacy of our 
biomarker for predicting susceptibility to hypoglycemia [e.g. aim 2 of the PI’s recently 
funded LA CaTS Roadmap Scholars grant (see appendix A below for project specific 
aims)]. Additionally, our use of continuous glucose monitoring (CGM) during the clamp 
procedure will provide novel data regarding the accuracy of CGM during hypoglycemic 
conditions in a controlled research setting.  
 
Given that the primary purpose of this study is for method development, there is no 
hypothesis being tested or statistical comparison being made across study participants.  
We will analyze the time course of the changes in our endpoints in each subject to verify 
that our procedure produces results that are similar to previously published data from 
other institutions which have used the hypoglycemic clamp technique.  This will allow us 
to develop an institutional MOP for the hypoglycemic clamp which can be used to safely 
implement this technique in future studies.  
 
 
Background 
Although Pennington Biomedical has extensive experience with euglycemic clamp 
procedures since its introduction by Dr. E Ravussin [e.g. 4, 5], experience performing 
the hypoglycemic clamp procedure is lacking. The procedural differences between the 
euglycemic clamp and hypoglycemic clamp are minimal.  For example, both procedures 
involve the paired infusion of insulin and glucose, with the rate of glucose infusion being 
adjusted to achieve a targeted steady-state blood glucose level.  In most instances, the 

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rate of insulin infusion is identical between the two procedures, whereas the rate of 
glucose infusion differs due to the difference in the targeted blood glucose 
concentration, which is approximately 100 mg/dL for the euglycemic clamp and 50 
mg/dL in the hypoglycemic clamp. Therefore, in order to achieve the lower steady state 
blood glucose, the initial rate of dextrose infusion during the first 45 minutes of the 
procedure will be significantly less than the typical rate of dextrose infusion during a 
euglycemic clamp.   
 
Given our considerable experience with euglycemic clamp and the similarities between 
the two procedures, we do not anticipate any issues preventing successful 
implementation of the hypoglycemic clamp procedure. In addition, we have consulted 
with Dr. Amir Moheet from the University of Minnesota who has directed multiple studies 
which utilize this procedure [e.g. 2, 6], to develop the procedure at Pennington (see 
attached letter of support). 
 
Study Participants 
We will enroll up to 8 healthy men or women (goal n=3 completers) in this method 
development study.  
 
Inclusion and Exclusion Criteria 
Inclusion Criteria: 
 Healthy male or female 
 Ages 18-40 years 
 BMI between 20 kg/m2 and 30 kg/m2 (±0.5 kg/m2 will be accepted) 
 Medically cleared for participation in the study  
 
Exclusion Criteria: 
 History of clinically diagnosed diabetes or a fasting blood glucose >126 mg/dL 
 Average screening blood pressure >140/90 mmHg 
 History of cardiovascular disease  
 Pregnant, planning to become pregnant, or breastfeeding 
 Based on the investigative team’s clinical judgement, a subject may not be 
appropriate for participation in the study. 
 
If eligible, individuals will be enrolled and undergo all testing procedures. 
 
Recruitment Methods 
Potential participants will be recruited through PBRC via IRB approved recruitment 
materials (e.g., landing page, listserv, social media). Individuals can either complete the 
webscreening form directly from the PBRC landing page, call PBRC directly, or e-mail 
the Recruitment Core. Potentially eligible participants will then undergo a phone screen 
to answer a series of yes or no questions regarding eligibility. Eligible individuals will be 
scheduled for a screening visit at PBRC.          
 
 
 

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Study Timelines 
A participant’s duration of study participation will be 3 days. The estimated duration to 
enroll all study subjects is anticipated to be four months. The estimated duration to 
complete the study is 6 months. 
 
Study Endpoints 
Primary endpoints:  
 Blood glucose levels 
 
Secondary endpoints: 
 Serum epinephrine, norepinephrine, glucagon, and cortisol. 
 CGM glucose 
 
The overarching goal of measuring these endpoints is to demonstrate our ability to 
safely perform hypoglycemic clamps, while simultaneously developing a systematic set 
of procedures (MOP) which will ensure a high rate of success and reliability in future 
studies utilizing this procedure. 
 
Procedures 
Participants will complete a screening visit, and one study visit conducted in the PBRC 
Inpatient Unit.  See Table 1 for schedule of assessments. 
 
Table 1. Schedule of Clinic Assessments 
 
 
Screening  
Visit  
Run-in 
Visit 
(2-3 days 
prior to 
Study Visit) 
Study 
Visit  
Procedures 
 
 
 
Informed Consent 
X 
 
  
Metabolic Weight, Height*, BMI 
X 
 
X 
Vital Signs (blood pressure, heart rate) 
X 
 
 X 
Fasting Blood Draw (CBC and Chem 14) 
X 
 
 
Screening Health Questionnaire 
X 
 
 
Medical History 
X 
 
 
Physical exam 
X 
 
 
Run-in meal 
 
X¥ 
 
CGM sensor placement 
 
X 
 
Pregnancy test (urine)  
 
 
 
X 
Hyperinsulinemic hypoglycemic clamp† 
 
 
X 
Continuous glucose monitoring 
 
 
X 
Meal (lunch) 
 
 
X 
*Height will only be measured during the Screening Visit 
¥Meal will be consumed the night before study visit.  Participant will pick up 
their meal up to 2-3 days prior to Study Visit. 
†IV procedure and serial blood collection 
CGM: Continuous glucose monitoring 

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Screening Visit:  
Participants will complete a screening visit at the Pennington Biomedical Outpatient 
Clinic to assess eligibility.  Participants will arrive in the morning to the PBRC Outpatient 
Unit, and after providing written informed consent, the following procedures will be 
completed: anthropometrics, vital signs, fasting blood draw (CBC and Chem 14), 
screening heath questionnaire, and medical history and physical examination by one of 
the medical staff members. The study MI, Dr. Hsia, will conduct a final review of the 
participants’ charts to determine eligibility.  
 
Run-in Visit: 
2-3 days prior to the study Visit, participants will be scheduled for a run-in visit to pick up 
their meal and a continuous glucose monitor (CGM) sensor will be placed on their 
abdomen. 
 
Study Visit:  
Eligible subjects will be scheduled for the Study Visit which will last approximately 8 
hours. Participants will be provided a run-in meal (supper) which will be consumed the 
evening before their visit. Participants will be instructed to 1) eat nothing but the meal 
provided after 3 PM, 2) eat the entire meal before 9 PM, and 3) drink only water after 3 
PM. Participants will be instructed to pick up their meal within 3 days prior to their study 
visit. 
 
Subjects will arrive at the inpatient unit in the morning after at least a 10 hour overnight 
fast.   Upon arrival to PBRC, participants will be admitted to the inpatient unit. Following 
measurement of metabolic weight, vitals, and pregnancy test (women only), the 4-hour 
hypoglycemic clamp procedure (30 minute baseline, 120 minute insulin infusion, and 90 
minute recovery) will be performed. Upon completion of the clamp procedure, 
participants will be provided lunch (Standard American Diet). Following lunch, the CGM 
sensor will be removed and pending a stable blood glucose level in the normal range, 
the participant will be discharged from the inpatient unit. This completes the study. 
 
Study Procedure Descriptions 
 Anthropometrics: Fasting body metabolic weight will be collected with participants 
wearing a hospital gown and underwear. Height will be collected once at 
screening.  
 
 Blood collection (study use): Approximately 7.5 mL of whole blood will be 
collected during the screening visit.  Approximately 120 mL of whole blood will be 
collected during the hypoglycemic clamp procedure for measurement of glucose, 
potassium, and counter-regulatory hormones.   The total volume of blood that will 
be collected during the study is approximately 128 mL.   
 
 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 

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in the abdominal area.  The sensor has a small needle-like probe that inserts into 
the subcutaneous fat of the abdomen and 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 via the blood glucose measurement obtained during the hypoglycemic 
clamp procedure. The CGM device records interstitial glucose every 5 minutes 
and will allow a more complete profile of blood glucose changes during the study 
visit. 
 
 Hyperinsulinemic-hypoglycemic clamp: An intravenous catheter will be placed in 
an antecubital vein for infusion of insulin and glucose.  A second catheter will be 
placed retrograde in a dorsal vein of the contra-lateral hand for blood withdrawal.  
The hand will be placed in a heating box or pad at 70°C for arterialization of 
venous blood.  A primed infusion of regular insulin (120 mU/min/m2) will be 
initiated and continued for approximately 2 hours.  Beginning 20 minutes prior to 
the start of the insulin infusion, arterialized venous blood glucose will be 
measured at 5 minute intervals via a Hemocue analyzer. Following initiation of 
insulin infusion, blood glucose will be allowed to fall to 50 mg/dL and then 
maintained at this level using a variable infusion of exogenous dextrose (20% 
solution). Our goal is to achieve steady-state (blood glucose stabilized at 50 
mg/dL) within the first 45 minutes following the start of insulin infusion, thus 
participants blood glucose will be maintained at this level for approximately 75 
minutes.   
 
In addition to the Hemocue glucose measurements, blood will be collected every 
15 minutes starting 15 minutes prior to insulin infusion, for determination of 
glucose, insulin levels, and counterregulatory hormone levels in circulation (10 
blood draws total). Potassium levels will be measured at three of these time 
points: -15, 60, and 120 minutes (relative to the start of the insulin infusion). See 
Table 2 for an overview of the timing of blood draws and measurements.  
 
Following discontinuation of the insulin, blood glucose will be normalized to 
baseline levels with the 20% dextrose infusion. The participant’s blood glucose 
will continued to be monitored every 15 minutes via Hemocue analyzer for 
approximately 90 minutes after discontinuation of insulin infusion to ensure 
baseline levels of blood glucose are achieved and maintained.  
 
If potassium levels are below 3.8 mmol/l at any of the three measurements (-15, 
60, or 120 minutes) a potassium infusion of 10 mEq/hr will be initiated and 
continued for the remainder of the procedure. In the event that a potassium 
infusion is initiated, potassium levels may be measured again following 
discontinuation of insulin infusion, i.e. during the 90 minute recovery period when 
blood glucose levels will be restored and maintained at baseline levels (120-210 
minutes relative to the start of the insulin infusion).  
 

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 Questionnaire:  Participants will complete a Screening Health Questionnaire (to 
assess general health) at the screening visit. 
 
 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. 
 
Power analysis 
Given that this is a method development study and we are not comparing across groups 
or individual participants, a power analysis was not performed. 
 
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: The purpose of this study is to demonstrate proficiency with the 
hypoglycemic clamp procedure.  This will involve demonstrating the ability to achieve 
and maintain a steady-state blood glucose of approximately 50 mg/dL for approximately 
75 minutes and that we are able to measure the known increases in counterregulatory 
hormone levels associated with exposure to hypoglycemia.  This will be indicated by an 
increase in all four hormones during the steady-state period of the procedure relative to 
baseline.  
 
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  
 Date of onset and date of resolution  
 Frequency (single / intermittent)  
 Maximum intensity (mild / moderate / severe)  
 Outcome (resolved / resolved with sequelae / not resolved) 

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 Action taken with respect to study drug/intervention (none / dose reduced / 
temporarily interrupted/ permanently discontinued/ intervention).  
 Withdrawal (yes / no) 
 Relationship to study drug/intervention 
 Whether the AE was “serious” or not (as defined below)  
 
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. 
 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 possibly 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.  

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Risks to Subjects 
This study involves major risk to study participants but with the possibility of benefitting 
the population at large.  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, 
Daniel Hsia, 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.  
 
 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. 
 
 Metabolic body weight: There is no risk to participants to have body weight 
measured. 
 
 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. 
 
 Hyperinsulinemic-hypoglycemic clamp: We expect participants to develop some 
symptoms of hypoglycemia during the procedure. Symptoms of hypoglycemia 
include: 
- Sweating,  
- Shakiness  
- Confusion 
- Increased heartbeat  
- feeling “low” 
These can be reversed within minutes by stopping the insulin infusion and by 
raising the blood sugar via increased rate of dextrose infusion. To be certain that 
the participant’s blood sugar level does not drop less than 30 mg/dL, blood 
glucose will be checked every 5 minutes and adjust as needed. If blood sugar 
drops too low, seizures or abnormalities in heart rhythm can occur. With careful 
monitoring, it is very unlikely that the participant’s blood glucose will drop to 

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unsafe levels. In the unlikely event that a participant’s blood glucose does drop to 
less than 30 mg/dL, dextrose will be administered immediately. 
 
The risks of hypoglycemia will be minimized by monitoring the blood glucose 
every 5 minutes and providing dextrose as needed to achieve the target level of 
blood glucose. Subjects will also wear a pulse oximeter during the study so we 
can monitor their heart rate. If we become unable to monitor the glucose during 
the study or if the participant develops a serious arrhythmia (as defined by the 
medical staff present), or if blood glucose drops to less than 30 mg/dL, the study 
will be stopped and dextrose will be given to normalize the blood glucose levels.  
Medical staff will be present throughout the procedure to monitor the patient. The 
study will be also be stopped if the medical staff feels that it is no longer safe for 
the participant to continue. With careful monitoring, it is very unlikely that a 
participant’s blood glucose will drop to less than 30 mg/dL. If blood glucose drops 
to less than 35 mg/dL, blood samples will be collected every 3 minutes. 
Additional countermeasures to hypoglycemia will also be utilized as necessary.   
    
 IV Procedure: There is a possibility of pain, bruising, or infection at the site of the 
needle insertion for the IV line. Trained personnel also minimize this risk.  
 
 Questionnaire:  There is a possibility that participants will be uncomfortable 
answering certain questions. Participants will be told that they may choose to 
skip any questions. 
 
Potential Benefits to Subjects 
There is no direct benefit to participants. 
 
Sharing of Results with Subjects 
Results will not be shared with participants.  
 
Setting 
This study will be conducted at Pennington Biomedical Research Center in the 
Outpatient Unit and Inpatient Unit.  
 
Resources Available 
Pennington Biomedical Outpatient and Inpatient staff are highly trained individuals with 
a breadth of experience in clinical research. All have completed human participants 
protection training and are highly knowledgeable of local study site and culture. A 
physician is 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.   
 
Compensation 

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Participants completing the study visit will receive $200. No compensation will be 
provided for screening or run-in visit.  
 
Confidentiality 
All data and specimens will be obtained solely for research purposes. These will include 
1) physical examinations, 2) medical history, 3) blood samples, and 4) adverse events 
during study participation. All data from individual participants 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 participants protection training. Data 
will be stored at Pennington Biomedical indefinitely. However, only study investigators, 
their staff, and collaborators will have access to the data. The study PI is responsible for 
receipt of the data.  
 
 
 
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 
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 

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discuss the study, its procedures and the participants’ options throughout study 
participation.  
 
References (bolded references attached with IRB submission). 
 
1. 
McDougal, D.H., et al., Glial acetate metabolism is increased following a 72-
h fast in metabolically healthy men and correlates with susceptibility to 
hypoglycemia. Acta Diabetol, 2018. 
2. 
Moheet, A., et al., Hypoglycemia-Associated Autonomic Failure in Healthy 
Humans: Comparison of Two vs Three Periods of Hypoglycemia on 
Hypoglycemia-Induced Counterregulatory and Symptom Response 5 Days 
Later. Journal of Clinical Endocrinology & Metabolism, 2014. 99(2): p. 664-
670. 
3. 
Seaquist, E.R., et al., Hypothalamic Glucose Transport in Humans During 
Experimentally Induced Hypoglycemia-Associated Autonomic Failure. The 
Journal of clinical endocrinology and metabolism, 2017. 102(9): p. 3571-
3580. 
4. 
Tam, C.S., et al., Defining Insulin Resistance From Hyperinsulinemic-Euglycemic 
Clamps. Diabetes care, 2012. 35(7): p. 1605-1610. 
5. 
Stull, A.J., et al., Skeletal Muscle Protein Tyrosine Phosphatase 1B Regulates 
Insulin Sensitivity in African Americans. Diabetes, 2012. 61(6): p. 1415-1422. 
6. 
Moheet, A., et al., Naltrexone for treatment of impaired awareness of 
hypoglycemia in type 1 diabetes: A randomized clinical trial. Journal of diabetes 
and its complications, 2015. 29(8): p. 1277-82. 
 
 
Appendix A- Project specific Aims of the PI’s recently funded LA CaTS Roadmaps 
Scholars grant. 
 
Specific Aims- 
 
Hypoglycemic complications are the major impediment to the maintenance of 
healthy plasma glucose levels in persons with diabetes. The applicant recently completed 
a LA CaTS pilot and feasibility project which identified a novel biomarker, glial acetate 
metabolism, which predicts susceptibility to hypoglycemia. By providing an assay to 
predict hypoglycemic complications, the development of this biomarker would 
revolutionize the treatment of persons with diabetes in a manner analogous to the HbA1c 
assay. This project will build on this initial study to facilitate the applicant’s 
competitiveness for extramural funding of this innovative line of research. 
 
Previous work established that hypoglycemic complications are associated with 
alterations in glial metabolism. Glia are the most abundant cell type in the brain and play 
an integral role in cerebral metabolism. Our prior work and that of others has generated 
three key observations which link alterations in the metabolic capacity of glial cells with 

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hypoglycemic exposure and hypoglycemic risk: 1) Exposure to hypoglycemia in both 
diabetic and non-diabetic subjects is associated with an increase in the ability of glial cells 
to metabolize acetate.  2) This effect is dose-dependent, i.e., greater exposure to 
hypoglycemia is associated with higher rates of glial acetate metabolism, and 3) 
Increases in glial acetate metabolism are correlated with acute susceptibility to 
hypoglycemia in both diabetic and non-diabetic subjects. Based on these observations, 
we hypothesize that alterations in glial acetate metabolism could be used as an effective 
biomarker for assessing the risk of hypoglycemic complications in individuals with 
diabetes.  
 
Securing extramural funding for the appropriate follow-up studies is presently 
limited due to a lack of preliminary data, the PI’s limited experience with clinical research, 
and the PI’s inexperience with key experimental techniques necessary to measure 
susceptibility to hypoglycemia. The overarching goal of this project is to overcome these 
limitations while developing fundamental knowledge regarding the relationship between 
glial metabolism and hypoglycemia. We will achieve this goal through the following 
specific aims: 
 
Aim 1: Establish the hyperinsulinemic-hypoglycemic clamp procedure at the Pennington 
Biomedical Clinical Trials Unit (PBRC CTU). This procedure is the gold standard for 
determining susceptibility to insulin-induced hypoglycemia and therefore is a necessary 
technique to extend the applicant’s preliminary findings and to compete successfully for 
extramural research support. Establishing this procedure will not only significantly 
advance the applicants research efforts, but it will also expand the scope of work available 
to all research projects at the PBRC CTU. The applicant will work with his mentoring team 
and existing collaborations with key personnel within the PBRC CTU to accomplish this 
aim.        
 
Aim 2: Determine if glial acetate metabolism predicts future susceptibility to 
hypoglycemia. We will measure glial acetate metabolism 7 days prior to exposure to 
insulin-induced hypoglycemia and determine how well this measurement predicts the 
magnitude of the counter-regulatory responses to a subsequent bout of hypoglycemia. 
Hypothesis: Glial acetate metabolism will be inversely proportional to the neuroendocrine 
response to hypoglycemia, that is, as glial acetate metabolism increases the 
neuroendocrine response will decrease.  This aim is a key test of our principal hypothesis. 
Establishing a link between glial acetate metabolism and future susceptibility to insulin-
induced hypoglycemia will significantly advance our initial findings while providing critical 
preliminary data to support future grant applications.