Clinical trial NCT00786825
Conditions: Type 1 Diabetes
Keywords: Type 1 diabetes Hypoglycemia unawareness
Sponsor: University of Minnesota
Start date: 2004-12-01; Completion date: 2017-12-01
Enrollment: 10 participants; Allocation: NON_RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
The long-range goal of this project is to determine the effects of diabetes and the hypoglycemic consequences of intensive therapy on in vivo brain glucose metabolism in humans. We will measure brain glycogen turnover and content in normal controls and subjects with diabetes under conditions of modest hyper-and hypoglycemia.

Detailed description:
On the morning before the study subjects will report to the Clinical Research Center and will be asked to stay at the Clinical Research Center for up to 72 hours. You will be asked not to eat anything after midnight. Small plastic catheters will be placed in two veins in subjects arms for infusion of glucose and for measuring blood glucose levels every 15-30 minutes. While subjects are in the study they will continuously receive a small dose of glucose enriched with 13C label either through the catheter or by drinking a small amount of a sugar water solution every 15-60 minutes. Subjects will be asked to transfer to the Center for Magnetic Resonance Research every 6-8 hours (except at night) where they will be asked each time to conduct an MRI scan as follows: Subjects will be placed on the patient bed in the magnet room for one-to-two hours. A nurse will stay with the subject at all times during this study. During some of the scans subjects may be asked to perform a visual task such as watching a changing checkerboard pattern through a mirror placed in front of their eyes. After each scan subjects will receive a low carbohydrate meal for weight-maintenance.

Subjects may be asked to receive an infusion of glucose and/or insulin one-to-three times before and/or after the infusion of 13C-enriched glucose starts. During these infusions the subject's blood sugar will be maintained either higher or lower than normal levels for up to 3 hours. This will be done to assess the effect of hyper- or hypoglycemia on brain glycogen metabolism.

Subjects may also be asked several days prior to the study to report to the Clinical Research Center for a "mock" study to see if they are comfortable with the MRI scan and/or to test their body's response to either protocol (as described above, but carried out at the Clinical Research Center). If subjects would like to try the MRI procedure in advance, this test will not exceed 2 hours. If we need to test the subject's response to the infusion, this "pre-test" will involve only drawing blood and administering glucose and will be performed as described for either protocol above, but will last not more than 4 hours.

Magnetic resonance measurements will be performed in the magnet and are very similar to magnetic resonance imaging exams (MRI). Subjects will always have the possibility to signal when they feel claustrophobic and wish to be removed from the magnet. The magnetic resonance measurements will be associated with a regular clunking noise that subjects may initially find unpleasant. For their comfort they will be offered to wear ear plugs. During the measurements in the magnet subjects will be asked to lie still for most of the time.

Arms: arm 1: Type 1 diabetes and Hypoglycemia unawareness arm 2: Healthy control subjects
Interventions: intervention 1: somatostatin
Intervention descriptions: intervention 1: Somatostatin may be used to suppress endogenous insulin secretion

Protocol documents:
Prot_SAP_001.pdf: 
Study Title: Measurement of Glucose/Glycogen Metabolism in Humans Using 
Magnetic Resonance at 4 or 7 Tesla   
 
PI: Elizabeth Seaquist 
 
NCT00786825 
IRB 9412M09040 
 
Revised: 9/30/2011 


SPECIFIC AIMS 
The fear of hypoglycemia and hypoglycemia unawareness limits the ability of patients with diabetes from 
achieving the level of glucose control known to reduce the risk of developing microvascular complications.  The 
long term goal of this project is to define the pathogenesis of hypoglycemia-associated autonomic failure 
(HAAF) with the aim of helping patients better manage their diabetes.  In HAAF, patients experience both 
hypoglycemia unawareness and defective glucose counterregulation.  Increased glucose availability to the 
brain is a likely contributor to the development of hypoglycemia unawareness either by increased glucose 
transport, decreased glucose utilization or increased glycogen storage following repeated episodes of 
hypoglycemia.  Work previously supported by this award provided evidence for elevated free glucose levels in 
cortical regions of patients with type 1 diabetes and hypoglycemia unawareness, but the impact of recurrent 
hypoglycemia on glucose levels and metabolism in the hypothalamus, the region in which both glucose 
sensing and coordination of the counterregulatory response are believed to occur, has not been evaluated. 
Growing evidence in animal models and humans also suggests that increased hypothalamic -aminobutyric 
acid (GABA) tone following recurrent hypoglycemia plays a central role in defective glucose counterregulation, 
but hypothalamic levels of GABA have not yet been measured in humans with and without HAAF.  We recently 
developed in vivo proton magnetic resonance spectroscopy (1H MRS) methodology to monitor glucose and 
GABA levels in the human hypothalamus at the ultra-high magnetic field of 7 tesla and to simultaneously 
measure all 4 kinetic parameters for cerebral glucose transport and utilization (KM transport, Vmax transport, KM 
utilization, Vmax utilization).  These methods will now be employed in humans together with the 13C-MRS 
protocol we have previously used to study brain glycogen to examine if changes in glucose availability (in the 
form of free glucose or glycogen) and GABA levels contribute to the development of hypoglycemia 
unawareness and defective glucose counterregulation in HAAF. 
The working hypotheses for this project are: 1) recurrent hypoglycemia, like that seen in subjects with type 1 
diabetes and iatrogenic hypoglycemia, will lead to an upregulation of glucose availability for the brain (by 
increased transport, and/or increased glycogen storage), thereby facilitating maintenance of cerebral 
metabolism during subsequent episodes of hypoglycemia, and 2) recurrent hypoglycemia will lead to an 
increased GABA tone in hypothalamus, thereby suppressing glucose counterregulation during subsequent 
episodes of hypoglycemia.  To address these hypotheses we will examine cerebral metabolism in subjects with 
type 1 diabetes and hypoglycemia unawareness, as well as in healthy volunteers preconditioned with 
euglycemia vs. recurrent hypoglycemia to induce HAAF, thereby permitting us to understand the clinical 
syndrome as well as isolate the effects of recurrent hypoglycemia from those induced by diabetes.  
Aim 1:  To determine the contribution of increased glucose availability in the brain to HAAF   
Specific Aim 1a: To measure kinetic parameters for glucose transport and metabolism in the hypothalamus of 
subjects with type 1 diabetes and hypoglycemia unawareness and in healthy volunteers preconditioned with 
euglycemia vs. recurrent hypoglycemia to induce HAAF.  
Hypothesis 1a.1:  Subjects with type 1 diabetes and hypoglycemia unawareness will have a higher Vmax 
transport than will healthy volunteers preconditioned with euglycemia.  
Hypothesis 1a.2:  Healthy volunteers will have a higher Vmax transport following preconditioning with recurrent 
hypoglycemia than with euglycemia.   
Specific Aim 1b: To measure brain glycogen turnover and content by 13C MRS in healthy volunteers 
preconditioned with euglycemia vs. recurrent hypoglycemia to induce HAAF.  
Hypothesis 1b:  Subjects will have higher glycogen content following preconditioning with recurrent 
hypoglycemia than with euglycemia. 
Aim 2:  To determine the contribution of increased GABA tone in the hypothalamus to HAAF   
Specific Aim 2: To measure euglycemic and hypoglycemic GABA levels in the hypothalamus of subjects with 
type 1 diabetes and HAAF and in healthy volunteers preconditioned with euglycemia vs. recurrent 
hypoglycemia to induce HAAF. 
Hypothesis 2a: Hypothalamic GABA levels will be lower during hypoglycemia than during euglycemia in both 
normal controls and patients with type 1 diabetes. 
Hypothesis 2b: Hypothalamic GABA levels will be higher both during euglycemia and during hypoglycemia in 
subjects with type 1 diabetes and HAAF than in matched controls preconditioned with euglycemia. 
Hypothesis 2c: Hypothalamic GABA levels will be higher both during euglycemia and during hypoglycemia in 
healthy volunteers following preconditioning with recurrent hypoglycemia than with euglycemia.  
Specific Aims
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Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


A. SIGNIFICANCE 
More than 25 million Americans have diabetes and hypoglycemia is a common event in the lives of 
patients who receive insulin or insulin secretagoges as therapy for their disease.  Inconvenient and disruptive 
to daily activities, it can also lead to loss of consciousness, seizures, and death.  Patients who experience 
recurrent episodes of hypoglycemia often develop hypoglycemia associated autonomic failure (HAAF); a 
clinical syndrome in which the first symptom of hypoglycemia is confusion or loss of consciousness (1).  The 
fear of HAAF prevents many patients with diabetes from achieving the glycemic goals known to reduce the risk 
of developing the microvascular complications of the disease (2).  Consequently, better ways to prevent HAAF 
are of critical importance in advancing care for patients with diabetes.  To achieve this goal first requires an 
understanding of the pathogenesis of this syndrome, which is the purpose of the project outlined in this 
application. 
Healthy humans have a robust and redundant counterregulatory response that prevents the development 
of hypoglycemia.  In patients with type 1 diabetes and advanced type 2 diabetes, this response is impaired (3).  
Such patients are unable to prevent a fall in glucose by reducing their own insulin secretion because of the 
beta cell failure associated with their disease.  They are also unable to release glucagon in response to 
hypoglycemia, likely due to a failure to reduce levels of insulin within the islet in response to the falling glucose 
(4).  They come to rely on hypoglycemia-induced secretion of epinephrine from the adrenal medulla and 
norepinephrine from the sympathetic nerves to cause the adrenergic symptoms typically associated with a low 
blood sugar to signal the need to ingest carbohydrates.  However, frequent episodes of hypoglycemia can lead 
to HAAF.  In HAAF, the blood glucose level that triggers catecholamine release is reduced into the range at 
which humans experience neuroglycopenia.  Patients with HAAF do not know they are experiencing 
hypoglycemia until they become confused or lose consciousness; a situation in which they cannot help 
themselves recover from the drop in blood sugar. 
The mechanisms responsible for the development of HAAF remain uncertain (5).  Clearly, upregulated fuel 
availability to the brain, either in the form of glucose (free or in glycogen reserves) or in the form of alternative 
fuels, can result in a failure to detect systemic hypoglycemia, the “unawareness” component of HAAF.  While 
there is conflicting data if global cerebral glucose transport or metabolism are altered in HAAF (6-8), regional 
alterations seem likely, particularly in the hypothalamus (9; 10), the region that senses systemic glucose and 
coordinates the counterregulatory response (11).  Our prior data using proton magnetic resonance 
spectroscopy (1H MRS) demonstrated higher cortical glucose levels in patients with type 1 diabetes and 
hypoglycemia unawareness than controls at the same plasma glucose levels (12). Subsequently, we found 
that oxidative glucose consumption in this brain region was not different in patients with type 1 diabetes and 
hypoglycemia unawareness than controls (13), indicating that the elevated glucose levels we observed were 
due 
to 
increased 
glucose 
transport, rather than decreased 
metabolism, 
and 
that 
such 
upregulated 
glucose 
transport 
may provide additional fuel to the 
brain 
during 
subsequent 
episodes of hypoglycemia to 
maintain normal function, at least 
for a while.  These studies had 
focused on the occipital cortex 
due to technical challenges with 
studying small and deep brain 
regions 
such 
as 
the 
hypothalamus.  In fact, glucose 
content and metabolism have not 
been measured simultaneously 
in 
the 
human 
hypothalamus 
before.  We have now overcome 
these challenges associated with 
acquiring MRS data from the 
human 
hypothalamus 
(14) 
(Appendix A, Preliminary studies) 
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Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


and further developed methodology to estimate kinetic parameters for glucose transport and utilization 
simultaneously (15) (Appendix B, Preliminary studies).  Therefore we are now in a unique position to address 
the affects of HAAF on glucose transport and metabolism in this brain region critical in CNS regulation of 
glycemic control.     
Regarding the second component of HAAF, the blunted counterregulatory hormonal response, recent 
investigations in rodents suggest a central role of an increased -aminobutyric acid (GABA) tone in the 
hypothalamus with recurrent hypoglycemia (16; 17).  A fall in the hypothalamic levels of GABA, the main 
inhibitory neurotransmitter in the brain, appears necessary for activation of glucose counterregulatory 
responses during hypoglycemia.  Recurrently hypoglycemic animals not only have elevated hypothalamic 
GABA levels, but they also fail to decrease these GABA levels in response to hypoglycemia (16).  
Furthermore, local application of glucose prevents this fall in a dose-dependent fashion, resulting in a positive 
correlation of extracellular glucose and GABA concentrations (17) and mechanistically linking increased 
glucose availability in this region to blunting of the GABA-regulated counterregulatory hormone release.  With 
recently developed technology (14) (Appendix A, Preliminary studies) we can now test if the same sequence of 
events occurs in the human brain.   
These prior data form the basis for our working hypotheses in the current application (Figure 1):  In the 
healthy brain, a drop in plasma glucose results in a drop in brain glucose, as well as a reduction in glycogen 
(18) and GABA (see Preliminary studies).  Following recurrent hypoglycemia, a drop in systemic glucose does 
not cause a similar drop in hypothalamic glucose levels due to upregulated transport capacity (
max
t
V
), and thus 
the signal that leads to glycogen mobilization and a fall in hypoglycemic GABA levels is reduced, thereby 
blunting the hypoglycemia induced sympathoadrenal response.  We will investigate these mechanisms both in 
healthy volunteers who have been preconditioned with recurrent hypoglycemia (a HAAF model), as well as in 
patients with type 1 diabetes, in order to distinguish the effects of HAAF alone from those of diabetes.  With the 
elucidation of the contributions of glucose (Aim 1a), glycogen (Aim 1b) and GABA (Aim 2) to the pathogenesis 
of HAAF, new knowledge will be available that will permit the development of better strategies to prevent and 
treat this condition, such as pharmaceutical interventions to prevent an increase of hypothalamic GABA levels.  
B. INNOVATION 
This project is innovative because it will use ultra-high field MRS to examine in vivo cerebral metabolism in 
humans with and without diabetes.  This is a significant advance because it will allow us to measure 
metabolites in a small, central structure like the hypothalamus, which is known to be critically important in 
recognizing and responding to hypoglycemia.  In our previous work, we performed 1H MRS at 4 tesla in the 
occipital cortex (12), but were unable to acquire sufficient signal in the hypothalamus to measure GABA and 
glucose concentrations in the hypothalamus.  As is demonstrated in a recent publication from our group (14) 
(Appendix A), this problem is overcome with the methodology developed for use at 7 tesla.  This technology 
will now allow us to test the elevated GABA tone hypothesis by direct measurements in the human 
hypothalamus for the first time. 
This project is also innovative because we will use the recently developed MRS methodology that allows 
for the simultaneous measurement of kinetic parameters for cerebral glucose transport and utilization (KM 
transport, Vmax transport, KM utilization, Vmax utilization) with dynamic measurements of brain glucose levels as 
a function of plasma glucose (15).  This methodology is a significant advance from previous investigations in 
humans which have relied on data collected under steady-state conditions and obtained kinetic parameters for 
glucose transport only by assuming a constant cerebral metabolic rate for glucose from literature.  Using the 
new 7 tesla MRS methods developed in our lab combined with this new modeling approach, we will be able to 
investigate glucose content and metabolism simultaneously in the human hypothalamus for the first time.  
C. APPROACH 
In this section we will first present preliminary data that demonstrates the feasibility of our experiments and 
then discuss the approach we will use to address each of our aims. 
C.1. PRELIMINARY STUDIES 
C.1.1. Methodology to create a human model of HAAF (Aims 1 and 2): 
Exposing healthy humans to recurrent hypoglycemia has repeatedly been shown to blunt the 
counterregulatory response to a subsequent episode of hypoglycemia (1; 2), just as observed in subjects with 
type 1 diabetes and HAAF (3).  An examination of a human model of HAAF will allow us to isolate the effects of 
recurrent hypoglycemia on brain metabolism from any additional effects of diabetes we may have observed in 
our previous experiments in subjects with type 1 diabetes and hypoglycemia unawareness.  To demonstrate 
Research Strategy                                                                                             Page 47
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


that we can create a human model of HAAF in our laboratory, four healthy humans participated in a two part 
study in which  hypoglycemic clamp studies were performed from 9-11 AM and 1-3 pm on day 1 and again 
between 9-11 AM on day 3 (n=2) or day 5 (n=2).  In each study, insulin was infused at a rate of 2.0 mU/kg/min 
for two hours and blood glucose was reduced to and then maintained at 50 mg/dl by the infusion of 20% 
dextrose, as guided by the measurement of blood sugars every 5 minutes.  Blood was collected every 30 
minutes during the clamp studies for the subsequent measurement of counterregulatory hormones in the 
Vanderbilt DRTC Core Laboratory.  We observed the epinephrine secretory response during the third episode 
to be blunted to 46 ± 8% of the response measured during the first episode.  
These data demonstrate that we can create a human model of HAAF in our laboratory and that healthy 
volunteers preconditioned with a repeated hypoglycemia protocol that involves 2 two-hour long hypoglycemic 
episodes on Day 1 have blunted counterregulatory response to hypoglycemia that lasts until Day 5 (critical for 
the design of the studies in Aim 1b).    
C.1.2. Methodology to measure kinetics of cerebral glucose transport and utilization (Aim 1a): 
The kinetic parameters for 
glucose transport and the rates 
of its metabolism in the brain 
have so far been obtained from 
separate 
experiments, 
for 
example 
measurements 
of 
brain glucose concentrations by 
1H MRS as a function of blood 
glucose to extract transport 
parameters (19; 20) and MR 
and PET tracer studies to 
estimate 
net 
glucose 
uptake/oxidation (10; 21).  In 
glucose 
transport 
measurements by 1H MRS, the 
kinetic parameters of cerebral glucose transport are traditionally obtained by fitting steady-state Michaelis-
Menten models to steady-state brain glucose concentrations obtained as a function of plasma glucose 
concentrations.  This way, KM for transport (
t
M
K
) and the ratio of Vmax for transport (
max
t
V
) to cerebral metabolic 
rate of glucose (CMRglc) are obtained and, assuming a constant CMRglc from prior work, 
max
t
V
 is calculated (19; 
20).  Such steady-state measurements do not take advantage of the additional information available in 
dynamic data.  The feasibility of determining all four kinetic parameters for cerebral transport and utilization 
(
t
M
K
, 
max
t
V
, 
util
M
K
, 
max
util
V
) by fitting both dynamic and steady-state data was demonstrated recently for acetate 
(22).  To apply the same approach to glucose transport and utilization, we measured brain and plasma glucose 
time courses during glucose infusions to raise and maintain plasma concentration at ~17 mmol/l for ~2 hours in 
5 healthy volunteers.  Occipital cortex glucose levels were measured from a 22-27mL voxel using previously 
described methods (12; 23) at 4 tesla.  We fitted a reversible, non-steady-state Michaelis-Menten model to 
these data (Fig. 2B) (15) combined with steady-state plasma vs. brain glucose concentrations taken from 
literature (Fig. 2A) (19; 20).  In addition to providing simultaneous measurements of glucose transport and 
utilization and obviating assumptions for constant CMRglc, this methodology does not necessitate infusions of 
expensive or radioactive tracers.  Using this new methodology, we found that the maximum transport capacity 
for glucose through the blood-brain barrier (
max
t
V
= 0.94 ± 0.21 μmol/g/min) was nearly two-fold higher than 
maximum cerebral glucose utilization (
max
util
V
= 0.48 ± 0.12 μmol/g/min).  Furthermore, the glucose transport and 
utilization parameters were consistent with previously published values for the occipital cortex.  These results 
were presented at a conference (15) and a manuscript describing them was submitted. 
These data demonstrate that the newly developed dynamic MRS methodology enables simultaneous 
measurements of the kinetic parameters for cerebral glucose transport and utilization (note that the steady 
state data of Fig. 2A will be obtained from the hypothalamus under the proposed studies by utilizing multiple 
hyperglycemic targets, unlike the preliminary studies in the occipital cortex that only utilized a single target, 
hence necessitating the use of literature data) and when tested in a widely studied region-of-interest, it 
provided transport parameters and a CMRglc consistent with previously published values obtained in 
independent studies.     
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Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


C.1.3. Methodology to measure glycogen turnover and content (Aim 1b): 
We have a long track record in measuring glycogen turnover and content using 13C MRS.  We developed 
the technology for applications in the human brain (24; 25), measured glycogen metabolic rate and content 
(26), demonstrated glycogen mobilization during hypoglycemia and increased glycogen synthesis following a 
single episode of moderate hypoglycemia in the healthy human brain (18).  In addition, we investigated 
glycogen content in the brains of patients with type 1 diabetes and hypoglycemia unawareness using the 
technology (see C.4. Progress Report).  Therefore we have extensive experience in investigating glycogen 
metabolism using 13C MRS in conjunction with IV infusions of 13C-glucose.      
C.1.4. Methodology to measure hypothalamic concentrations of GABA and glucose (Aims 1a and 2): 
We have recently developed MRS methodology to obtain 
spectra of high quality (good signal-to-noise ratio and 
resolution) from any region in the cerebrum, cerebellum and 
brainstem at 7 tesla (14).  We applied this technology to 
obtain neurochemical profiles, including GABA and glucose, 
from the human hypothalamus.  GABA levels were quantified 
with Cramér-Rao lower bounds (CRLB, estimated errors 
(%SD) of metabolite quantification calculated by the 
quantification software LCModel) of 15% in these spectra 
obtained 
over 
10 
minutes 
at 
euglycemia 
(Fig. 
3).  
Hypothalamic GABA levels were estimated at 3.5-4 µmol/g at 
euglycemia (Fig. 4), i.e. 3-4 fold higher than cortical GABA 
concentrations (27), in excellent agreement with biochemical 
literature (28).  Glucose levels were quantified with CRLB of 
33%, which was similar to the CRLB obtained at the 
euglycemic baseline measurements of cortical glucose levels 
at 4 tesla (Fig. 2B).  Therefore these data demonstrate that 
we will be able to obtain kinetic parameters for glucose transport and utilization in the hypothalamus with the 
same reliability (measurement errors) as we have in the occipital cortex.  
The CRLB are a good estimate of test-retest reproducibility of metabolite measurements, however we also 
obtained test-retest data from the hypothalamus at euglycemia in a healthy volunteer at 7 tesla (Fig. 4A, green 
trace).  The coefficient-of-variance for the GABA measurement was 3% and for glucose 20% in this study.  We 
further investigated the response of both neurochemicals to hypoglycemia in a patient with type 1 diabetes and 
hypoglycemia unawareness and an age-, gender- and BMI-matched healthy volunteer (Fig. 4).  The reliability 
of the glucose measurement decreased as the hypothalamic glucose levels dropped, therefore we will conduct 
the glucose transport and metabolism studies by employing hyperglycemic targets as has been traditionally 
done to study cerebral glucose transport by MR (15; 19; 20).  On the other hand, GABA levels were reliably 
quantified throughout the hypoglycemic clamp and we observed a decrease in GABA levels in response to 
hypoglycemia in both the healthy volunteer and the patient (Fig. 4).  However, the GABA decrease from 
baseline to the last data point 
was 36% in the case of the 
healthy volunteer while it was 
26% in the case of the patient. 
These data demonstrate our 
ability 
to 
reliably 
measure 
GABA 
and 
glucose 
concentrations 
for 
the 
proposed studies in Aims 1a 
and 
2 
and 
support 
our 
hypothesis 
regarding 
an 
attenuated 
hypothalamic 
GABA 
response 
to 
hypoglycemia 
in 
type 
1 
diabetes 
and 
hypoglycemia 
unawareness.   
Research Strategy                                                                                             Page 49
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


C.2. RESEARCH DESIGN 
Aim 1a: To measure kinetic parameters for glucose transport and metabolism (KM transport, Vmax 
transport, KM utilization, Vmax utilization) in the hypothalamus of subjects with type 1 diabetes with 
hypoglycemia unawareness and in healthy volunteers preconditioned with euglycemia vs. recurrent 
hypoglycemia to induce HAAF.  
In these experiments, subjects with type 1 diabetes with hypoglycemia unawareness as well as normal 
controls, preconditioned on separate occasions with euglycemia and with recurrent hypoglycemia, will have 
hypothalamic glucose concentrations measured continuously using 1H MRS at 7 tesla over a ~1.5 hour period 
during which blood glucose concentrations will be experimentally increased from 95 mg/dl to one of 3 target 
hyperglycemic levels (200 mg/dl (11 mM), 300 mg/dl (16.5 mM), 400 mg/dl (22 mM)). 
Study rationale: 
We have previously demonstrated that steady state glucose concentrations in the occipital cortex are 
higher in subjects with type 1 diabetes and hypoglycemia unawareness than in matched controls studied under 
the same conditions (12), with no differences in oxidative glucose consumption (13).  We have interpreted 
these data to suggest that brain glucose transport is upregulated in response to recurrent hypoglycemia and 
have proposed that this is one mechanism that contributes to the development of HAAF.  However, these 
observations do not provide evidence of how recurrent hypoglycemia alters glucose transport or metabolism in 
the hypothalamus.  In addition, the finding of higher steady state brain glucose concentrations in subjects with 
type 1 diabetes and hypoglycemia unawareness than in controls does not necessarily mean that this is purely 
a result of recurrent hypoglycemia, and not a confound of diabetes.  To overcome these limitations, we will now 
directly examine hypothalamic glucose transport and metabolism by utilizing dynamic measurements of brain 
glucose levels as a function of plasma glucose.  Such an approach will permit the simultaneous measurement 
of KM transport, Vmax transport, KM utilization, Vmax utilization in this critical region.  We have selected 3 
hyperglycemic targets to obtain steady-state brain vs. plasma glucose concentrations over a wide range of 
plasma glucose concentrations (as shown for occipital cortex in Fig. 2A).  We will study subjects with type 1 
diabetes and hypoglycemia unawareness, as well as a human model of HAAF.  Note that we do not use the 
term “HAAF” in the case of the patients since we will not perform a hypoglycemic clamp in patients as part of 
this aim to confirm blunted counterregulatory response prior to the MR scan; however the presence of HAAF 
has been repeatedly demonstrated in subjects with type 1 diabetes and hypoglycemia unawareness (12), as 
assessed by a standardized questionnaire we will use. If healthy humans demonstrate an increase in the Vmax 
transport in the hypothalamus following the induction of HAAF, it will provide firm support that this mechanism 
participates in the development of the syndrome. 
Research Subjects: 
Subjects with and without diabetes will be recruited from a registry maintained by the investigators of 
individuals interested in participating in this type of research.  In addition, subjects with diabetes will be 
recruited from the Endocrine Clinic at the University of Minnesota and healthy controls will be recruited from 
the University of Minnesota community.  Inclusion criteria for the subjects with diabetes include well controlled 
type 1 diabetes (hemoglobin A1C <7.5%), frequent episodic hypoglycemia (at least 2 episodes per week for 
the preceding two months) and hypoglycemia unawareness verified by a standardized questionnaire (29).  
Healthy controls will be age (within 5 years), gender, and body mass index (within 4 kg/m2) matched to the 
subjects with diabetes.  Exclusion criteria for both groups of subjects include history of stroke, seizures, 
neurosurgical procedures, or arrhythmias, and use of drugs that can alter glucose metabolism (other than 
insulin for the patients with diabetes).   Subjects must also meet requirements for a study in the magnet, which 
includes weight less than 300 lbs and the absence of metallic substances in their body.  After screening and 
consent, each patient will be randomized to one of three glucose targets (200, 300, 400 mg/dl); the patient’s 
matched control will be assigned to the same target. 
Study Protocol: 
Subjects with type 1 diabetes and hypoglycemia unawareness will report to the Center for Magnetic 
Resonance Research (CMRR) in the morning in the fasting state.  They will be instructed to manage their 
diabetes during the day prior to the study in such a way as to minimize the amount of subcutaneous insulin 
present during the experiment while maintaining adequate control.  Upon arrival, an intravenous catheter will 
be placed antegrade in a forearm for subsequent infusions and a catheter will be placed retrograde in the lower 
leg for blood sampling.  An intravenous infusion of insulin will then be started and adjusted as necessary to 
bring blood glucose to 95 mg/dl.  Blood will be collected every 10 minutes for measurement of glucose on a 
Research Strategy                                                                                             Page 50
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


nearby Analox machine to guide adjustments in the 
insulin infusion rate.  The experiment will begin when 
the subject is at 95 mg/dl.  At time 0, the intravenous 
insulin infusion will be fixed at 0.5 mU/kg/min, samples 
for blood glucose will be collected every 5 minutes and 
an intravenous infusion of glucose (20% dextrose) will 
be administered as necessary to maintain blood 
glucose at 95 mg/dl.  At +5 minutes, subjects will be 
placed into the 7 tesla magnet (Fig. 5).  Blood glucose 
will be maintained at 95 mg/dl during the next 25 
minutes as the hypothalamic volume-of-interest (VOI) is 
selected and baseline MRS data are acquired.  At + 30 
min, subjects will be given an intravenous bolus 
injection of glucose over 1-2 minutes using the formula of administering 2 mg glucose/kg body weight for each 
1 mg/dl increase desired that we have used with success previously (19).  Immediately after the bolus 
injection, a continuous infusion of 20% dextrose will be started and the rate of administration will be adjusted to 
maintain the desired glucose target of 200 mg/dl, 300 mg/dl, or 400 mg/dl.  MRS data will be collected 
continuously starting 10 minutes before the bolus injection (baseline) until the end of the study ~60 minutes 
after the bolus, which in our experience is sufficient to reach steady-state glucose concentrations in the brain 
and maintain them for at least 20 minutes (Fig. 2B).  At the completion of the study, the infusions will be 
discontinued; the subject will resume their usual insulin regimen, and be fed a meal.  
Healthy controls will be studied both after preconditioning with euglycemia and after preconditioning with 
recurrent hypoglycemia to induce HAAF, done in random order separated by a minimum of 6 weeks (Fig. 6).  
Female controls will have their two studies two months apart in order to control for phase of menstrual cycle.  
We will follow a hypoglycemia preconditioning protocol we (see preliminary data) and others (30; 31) have 
found to induce HAAF.   
For preconditioning, subjects will undergo 
hyperinsulinemic clamp studies between 8-10 
AM on Day 1, 2-4 PM on Day 1, and 8-10 AM on 
Day 2.  For hypoglycemia preconditioning, the 
target glucose during the clamp studies will be 
50 mg/dl and for euglycemia preconditioning the 
target glucose will be 95 mg/dl. For each clamp 
study, subjects will be given intravenous insulin 
at a rate of 2.0 mU/kg/min for two hours and 
blood glucose will be maintained at target by the 
infusion of 20% dextrose, the rate of which will 
be adjusted based on blood glucose values 
measured on the Analox from samples collected 
every 5 minutes.  Samples will also be collected 
every 30 minutes for later measurement of 
glucagon, catecholamines, cortisol, and growth 
hormone.  Potassium phosphate will be infused 
at 4 mEq/hour as long as the insulin is being 
infused.  At the completion of the two hour 
period, subjects will be given glucose as needed 
to restore euglycemia and then allowed to rest 
for two hours.  On Day 2, subjects will go to the 
CMRR in the afternoon for assessment of hypothalamic glucose transport and metabolism using 1H MRS.  
Upon arrival, an intravenous catheter will be placed antegrade in a forearm for subsequent infusions and a 
catheter will be placed retrograde in the lower leg for blood sampling.  Subjects will be placed into the 7 tesla 
magnet and MRS data will be acquired from the hypothalamus with the same protocol as that for patients (Fig. 
5).  At + 30 min, subjects will be given a intravenous bolus injection of glucose over 1-2 minutes using the 
formula of administering 2 mg glucose/kg body weight for each 1 mg/dl increase desired that we have used 
with success previously (19).  Following the bolus injection, the desired glucose target of 200 mg/dl, 300 mg/dl, 
or 400 mg/dl will be maintained with a continuous infusion of 20% dextrose at a varying rate based on glucose 
Research Strategy                                                                                             Page 51
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


levels obtained in blood samples every 5 minutes. Insulin will not be administered due to the endogenous 
production of insulin in healthy volunteers and because plasma insulin concentrations up to 100 mU/l do not 
change brain glucose concentrations (32).  At the completion of the study, the infusions will be discontinued 
and subjects will be fed a meal. 
1H MRS methods: 
Studies will be performed on a 7T scanner operated with a Siemens console, using a 16-channel 
transmit/receive phased array coil (33).  Methods to acquire the 1H MRS data (including evaluation of the CSF 
contribution to each VOI) will be identical to those described before, using short echo (TE = 8 ms), single voxel 
stimulated echo acquisition mode (STEAM) spectroscopy (14, Appendix A, 34).  Power deposition in each VOI 
will be optimized by B1 shimming in each subject as described before (14; 35).  Methods to quantify the 
neurochemical profiles using LCModel (36), and to assess the reliability of metabolite concentrations based on 
Cramér-Rao lower bounds, will be identical to those described before (14; 34; 37).   
Modeling methods: 
A kinetic model of glucose transport through the blood-brain-barrier (BBB) via reversible symmetric 
Michaelis-Menten kinetics and irreversible utilization in brain tissue will be utilized (15).  In this model, the time-
dependent brain glucose concentration 
brain
Glc
is given by  
 
         with   
 
 
where
t
M
K
 is the Michaelis-Menten constant for transport of glucose through the BBB, 
max
t
V
 is the maximal 
transport rate of glucose, 
d
V  is the physical distribution volume for brain glucose (0.77 ml/g) (38; 39) and 
util
M
K
 
and 
max
util
V
 are the Michaelis-Menten constant and the maximal rate for glucose utilization.  CMRglc is the 
cerebral metabolic rate for glucose consumption and is modeled by irreversible Michaelis-Menten kinetics.  
The four unknown parameters in these equations are
t
M
K
, 
max
t
V
 and 
util
M
K
 and 
max
util
V
 and the measured data sets 
are 
plasma
Glc



 and 

brain
Glc
under steady–state conditions and as a function of time in dynamic experiments.  
Simplex, Broyden-Fletcher-Goldfarb-Shanno and Levenberg-Marquart algorithms will be used to extract kinetic 
parameters by minimization of the cost function as defined by the least-square difference between model 
simulation and experimental data.  
Sample size justification and data analysis plan: 
max
t
V
 will be compared between patients and euglycemia conditioned controls with a paired t-test; if data 
are highly skewed, non-parametric alternatives will be considered.  Similar comparisons will be carried out, 
within the controls, between the results following euglycemic preconditioning and following hypoglycemic 
conditioning.  Pilot data standard deviation in 
max
t
V
 (section C1.2) indicate that with 12 participants per group 
we have 85% power to detect paired-group differences of 14% in 
max
t
V
 with type I error of 5%.  We have 
budgeted for 15 per group to allow for some failed scans without sacrificing power.  Our previous data in the 
occipital cortex demonstrated ~17% higher glucose concentrations in subjects with type 1 diabetes and 
hypoglycemia unawareness vs. controls.  Therefore, we expect to be able to detect a similar effect size in the 
hypothalamus in the proposed studies with 12 subjects in each group.  While our power calculations are based 
on SD in 
max
t
V
 because we hypothesize that unidirectional glucose transport is upregulated in HAAF, our 
studies will also be able to reveal a change in 
max
util
V
, if HAAF involves a change in glucose metabolism, thanks 
to the simultaneous measurement of these parameters with the new modeling approach.   
Potential pitfalls and solutions: 
Data will be collected as blood glucose is experimentally increased from 95 to 200, 300, or 400 mg/dl and 
we assume that the same kinetic constants will apply under hypoglycemia conditions.  Under Aim 2, we will 
also obtain hypothalamic glucose data under hypoglycemia because we will be using the same MRS methods 
to obtain neurochemical profiles in these two aims.  Since these data will also be available, we will assess the 
reliability of hypothalamic glucose measurements at hypoglycemia at a lower time resolution than that shown in 
Fig. 2B, e.g. in spectra acquired every 10-20 minutes instead of every 2.5 min.  Because we save our MRS 
data in single shots, we can process these data to add over different time windows.  Indeed, such lower time-
max
max
[
]
[
]/
[
]
[
]
[
]/
t
t
plasma
brain
d
brain
glc
t
M
plasma
brain
d
V
Glc
V
Glc
V
d Glc
CMR
dt
K
Glc
Glc
V





max[
]
[
]
util
brain
glc
util
M
d
brain
V
Glc
CMR
K
V
Glc



Research Strategy                                                                                             Page 52
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


resolution data may also provide hypoglycemic glucose concentrations in hypothalamus.  However, we chose 
to design the primary experiments under Aim 1a with hyperglycemic targets which we know will provide us with 
the kinetic parameters with certainty.    
Another potential problem we may encounter is that the protocol we use to induce HAAF will not be 
effective for all subjects enrolled in the study.  To ensure that HAAF is present at the time we do our 
experiment, we will measure counterregulatory hormones in blood collected from subjects during the first and 
third hypoglycemic clamps done before the MRS experiment.  If the epinephrine response (the hormone most 
blunted during most protocols in which HAAF is induced in humans (30; 31)) during the third hypoglycemic 
clamp is > 70% of the response measured during the first hypoglycemic clamp, we will conclude that HAAF 
was not induced and the data will not be used for analysis.  If HAAF fails to be induced in 1/3, 2/5, 3/7, 4/9,… 
of the control studies (we will monitor in an ongoing way), we will change the protocol to induce HAAF to 
include more episodes of hypoglycemia before the MRS experiment.   
Aim 1b:  To measure brain glycogen turnover and content by 13C MRS in healthy volunteers 
preconditioned with euglycemia vs. recurrent hypoglycemia to induce HAAF.  
In these experiments, the impact of recurrent hypoglycemia on brain glycogen turnover and content will be 
measured using 13C MRS in healthy humans. 
Study rationale: 
By monitoring incorporation of intravenously infused 13C-glucose into glycogen by 13C MRS, we have 
previously demonstrated increased brain glycogen synthesis following a single episode of moderate 
hypoglycemia in healthy humans, indicating glycogen supercompensation (18).  On the other hand, we found 
that glycogen content in subjects with type 1 diabetes and hypoglycemia unawareness is not higher than 
controls (See C.4. Progress Report).  We have interpreted these findings to indicate that supercompensated 
glycogen levels do not contribute to the pathogenesis of HAAF, perhaps because upregulation of glucose 
transport in these subjects ensures that they do not experience the reduction in brain glucose content 
necessary to stimulate glycogen supercompensation following the modest levels of hypoglycemia that routinely 
occur.  However, it is also possible that diabetes itself alters brain glycogen metabolism in such a way as to 
confound the response to recurrent hypoglycemia.  Therefore, we intend to examine the impact of recurrent 
hypoglycemia on brain glycogen metabolism in healthy volunteers without the confounding effects of diabetes.  
We will monitor 13C-glucose incorporation into glycogen for up to 80 hours with the goal of turning over the 
entire glycogen pool for which shorter 13C label infusion periods have been insufficient (18; 26).  If we find that 
brain glycogen content is higher after induction of HAAF in normal volunteers, we can conclude that diabetes 
does alter brain glycogen metabolism in ways independent of recurrent hypoglycemia.  If we find that brain 
glycogen content is the same or lower after induction of HAAF, we can conclude that supercompensated 
glycogen does not contribute to the development of HAAF. 
Research Subjects: 
Healthy controls will be recruited from the University of Minnesota community.  Exclusion criteria will 
include history of stroke, seizures, neurosurgical procedures, or arrhythmias, and use of drugs that can alter 
glucose metabolism.  Subjects must also meet requirements for a study in the magnet, which includes weight 
less than 300 lbs and the absence of metallic substances in their body.  
Study Protocol: 
We will examine the impact of repeated episodes of hypoglycemia on brain glycogen content and turnover 
in healthy controls.  As in Aim 1a, subjects will be studied both after preconditioning with euglycemia and after 
preconditioning with recurrent hypoglycemia to induce HAAF, done in random order separated by a minimum 
of 6 weeks (Fig. 6).  Female controls will have their two studies two months apart in order to control for phase 
of menstrual cycle.   
The hypoglycemia preconditioning and euglycemia preconditioning protocols will be identical to those 
described in Aim 1a, with the first MRS session to measure 13C-glycogen level in the afternoon on Day 2 (Fig. 
6).  At minute +30 following the end of the last hypoglycemic or euglycemic clamp, a bolus injection of 20 g of 
[1-13C]glucose (prepared as 20% weight/volume D-glucose in water with 50% isotopic enrichment) will be 
administered to subjects regardless of their preconditioning protocol.  Twenty minutes later a continuous 
infusion of 2 mg/kg/min of [1-13C]glucose (prepared as 20% weight/volume D-glucose in water with 25% 
isotopic enrichment) will  be started and then adjusted to maintain blood glucose at ~125 mg/dl based on 
samples collected every 10-60 minutes and measured on an automatic glucose analyzer.  The infusion of 13C-
glucose will continue for ~82 hours.  Cerebral 13C-glycogen levels will be measured at ~5, 12, 22, 34, 46, 58, 
Research Strategy                                                                                             Page 53
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


70 and 82 hours after the end of the last hypoglycemic or euglycemic clamp.  Blood samples will also be frozen 
for the later determination of plasma insulin concentrations by a chemiluminescent assay (Immulite, Diagnostic 
Products Corporation, Los Angeles, CA), and of the isotopic enrichment of the plasma glucose by gas-
chromatography-mass spectroscopy (GC-MS) as described previously (40). 
13C MRS methods: 
13C-glycogen levels in the brain will be measured on a 4 T / 90 cm magnet (Oxford/Varian) using a 
quadrature 14 cm 1H surface coil combined with a 9 cm diameter linear 13C coil (41).    The [1-13C]glycogen 
NMR signal will be localized in a 7 × 5 × 6 cm3 voxel in the occipital lobe as described before (25).  The 
amount of 13C label in the C1 position of glycogen will be quantified by the external reference method (23; 24).  
The [1-13C]glycogen concentrations will be divided by the plasma glucose isotopic enrichment (IE) to correct for 
differences in IEs between subjects and to determine the newly synthesized glycogen concentrations.  13C-
glycogen levels will be corrected for the cerebrospinal fluid (CSF) content of the voxel (34). 
Sample size justification and data analysis plan: 
13C-glycogen levels will be compared following euglycemic preconditioning to following hypoglycemic 
conditioning with a repeated measures model with effects for precondition, acquisition time (5, 12, etc. hours), 
and their interaction; we will also test for an effect of the order of preconditioning. Correlation structures 
appropriate for doubly-repeated measures (across acquisition times within preconditioning, for two 
preconditioning sessions) will be used. Previous work (18) shows standard deviations of the group difference 
ranging from 0.2 to 0.4 µmol/g across the acquisition times, indicating that with 5 participants we will have 85% 
power to detect paired-group differences between 0.36 and 0.73 µmol/g with type I error of 5%. Per-
acquisition-time group comparisons will be adjusted for multiple comparisons using Sidak’s method. We have 
budgeted for 6 participants to allow for one failed study without sacrificing power.  Since we have detected 
highly significant differences during and following single hypoglycemia vs. euglycemia in our prior studies with 
5 subjects per group (18), we expect to be able to also detect a difference with 5 subjects who will serve as 
their own controls, if recurrent hypoglycemia does affect glycogen content/metabolism. 
Potential pitfalls and solutions: 
We have had success in recruiting subjects to participate in similar protocols in the past (18; 26) and all of 
the methods are well established in our laboratory.  Thus, we do not anticipate any problems with the 
completion of the work.  As in our previous work (18; 24; 26) glycogen content and turnover will be studied in 
the occipital lobe because of the low cerebral glycogen concentrations, the large size of the molecule that 
broadens its MR signal and the resulting small 13C MRS signal.  It would be ideal to study hypothalamic 
glycogen levels and turnover; however this is not possible with the currently available technology and no other 
methods exist to monitor glycogen in the intact human brain.     
As for aim 1a, we may find that the protocol we use to induce HAAF will not be effective for all subjects 
enrolled in the study.  To ensure that HAAF is present at the time we examine glycogen metabolism, we will 
measure counterregulatory hormones in blood collected from subjects during the hypoglycemic clamps.  If the 
epinephrine response (the hormone most blunted during most protocols in which HAAF is induced in humans 
(30; 31)) during the third hypoglycemic clamp is > 70% of the response measured during the first hypoglycemic 
clamp, we will conclude that HAAF was not induced and the data will not be used for analysis.  If HAAF fails to 
be induced in 1/3, 2/5, 3/7, 4/9,… of the studies (we will monitor in an ongoing way), we will change the 
protocol to induce HAAF to include more episodes of hypoglycemia before the glycogen experiment. 
Aim 2: To measure euglycemic and hypoglycemic GABA levels in the hypothalamus of subjects with 
type 1 diabetes and HAAF and in healthy volunteers preconditioned with euglycemia vs. recurrent 
hypoglycemia to induce HAAF. 
In these experiments, subjects with type 1 diabetes and HAAF (as confirmed by counterregulatory 
hormone measurements during a hypoglycemic clamp), as well as normal controls with and without HAAF, will 
have hypothalamic GABA levels measured at a blood glucose of 95 mg/dl and again at a glucose level of 50 
mg/dl by 1H MRS at 7 tesla over a ~one hour period. 
Study rationale: 
Recent studies in animals suggest that hypothalamic GABA may be important in regulating the 
counterregulatory response to hypoglycemia and that elevated GABA levels may contribute to the 
development of HAAF (17).  Recurrent hypoglycemia leads to increased basal GABA inhibitory tone in the 
ventromedial hypothalamus (VMH) and suppresses the counterregulatory response to subsequent episodes of 
hypoglycemia (16).  The role of GABA in regulating the counterregulatory response and in the development of 
Research Strategy                                                                                             Page 54
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


HAAF in humans is unknown.  Therefore, we will use our 
newly 
developed 
1H 
MRS 
methods 
to 
measure 
hypothalamic GABA in subjects with type 1 diabetes and 
HAAF and in healthy controls preconditioned with 
euglycemia vs. recurrent hypoglycemia to induce HAAF. 
We predict that hypothalamic GABA will be lower during 
hypoglycemia than euglycemia in controls and subjects 
with type 1 diabetes, that induction of HAAF will lead to an 
increase in hypothalamic GABA under both euglycemic 
and hypoglycemic conditions, and that subjects with type 1 diabetes and HAAF will have higher levels of 
hypothalamic GABA under both euglycemic and hypoglycemic conditions than euglycemia preconditioned 
controls (Fig. 7).  The MRS acquisition protocol will also provide concentrations of the precursor of GABA, 
glutamate (Fig. 1), which will potentially provide supporting information about GABA metabolism, however our 
hypotheses are based on GABA levels, which have been reported to change in prior animal work.   
Research Subjects: 
Recruitment strategies and inclusion and exclusion criteria will be identical to those in Aim 1a, except we 
will also exclude subjects who take drugs that can alter GABA metabolism (such as benzodiazepines). 
Study Protocol: 
Subjects will report to the Center for Magnetic Resonance Research in the morning in the fasting state. 
Subjects with diabetes will be instructed to manage their diabetes in such a way as to minimize the amount of 
subcutaneous insulin present at the time of study while maintaining adequate control.  Healthy subjects will 
serve as their own controls and be studied following the Day 1 hypoglycemia and euglycemia preconditioning 
protocols described for Aim 1 (Fig. 8).  At least six weeks will separate the two studies, which will be done in 
random order.  Female controls will have their two studies two months apart in order to control for phase of 
menstrual cycle. 
On the day of the MRS experiment, an intravenous 
catheter will be placed antegrade in a forearm for 
subsequent infusions and a catheter will be placed 
retrograde in the lower leg for blood sampling. Patients 
with diabetes will be started on a variable rate infusion of 
intravenous insulin designed to bring their blood glucose 
to 95 mg/dl.  To adjust the rate, blood will be collected 
every 5 minutes and tested on a nearby Analox machine. 
The experiment will begin, for both patients and controls, 
when the subject is at euglycemia.  At time 0, insulin will 
be started at a rate of 2.0 mU/kg/min, potassium 
phosphate will be started at a rate of 4 mEq/hr, and 
glucose (20% dextrose) will be administered at a variable 
rate to maintain the blood glucose at 95 mg/dl.  Subjects 
will then be placed into the 7 tesla magnet and 
hypothalamic GABA will be measured.  At +60, blood 
glucose will be reduced to 50 mg/dl.  The time course for 
hypothalamic GABA will be measured as shown in Fig. 4. 
Samples for the measurement of glucagon, epinephrine, norepinephrine, cortisol, and growth hormone will be 
collected at minutes 0, 60, 90, and 120.  At the completion of the measurement, subjects will be taken out of 
the magnet, blood glucose will be restored, and subjects will be fed. 
1HMRS methods: 
Spectra will be acquired and quantified using identical methods to those described in Aim 1a.  We chose 
to utilize STEAM spectroscopy to quantify GABA levels, as opposed to edited spectroscopy, since the GABA 
quantification using STEAM was validated against edited spectroscopy (42) and STEAM provides better 
signal-to-noise and additional metabolite concentrations such as glutamate (the precursor of GABA) and 
glucose (see Potential pitfalls for Aim 1a). 
Sample size justification and data analysis plan: 
Research Strategy
Page 55
Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.


Euglycemia vs. hypoglycemia states:  After preconditioning with euglycemia among the controls, GABA 
levels will be compared during euglycemia to during hypoglycemia with a repeated measures model with 
effects for glycemic state, acquisition time (5, 15, etc. minutes, Fig. 4), and their interaction. Correlation 
structures appropriate for doubly-repeated measures (across acquisition times within glycemic states, for two 
glycemic states) will be used. These comparisons will be repeated among the controls after preconditioning 
with hypoglycemia, and among the patients. Pilot work (Fig. 4) shows a standard deviation of the control group 
difference between euglycemic states and hypoglycemic states of 0.52 µmol/g, indicating that with 10 
participants we have 85% power to detect paired-group differences of 0.53 µmol/g with type I error of 5%.  In 
the case that standard deviations are twice those observed in Figure 4, we have 85% power to detect 
differences of 1.10 µmol/g. Per-acqusition-time glycemic state comparisons will be adjusted for multiple 
comparisons using Sidak’s method. We have budgeted for 12 participants to allow for two failed scans without 
sacrificing power.  Patients vs. controls: Similar comparisons will be carried out between GABA levels in 
patient euglycemia states and in control euglycemia states after the controls are euglycemic preconditioned. 
This will be repeated for GABA in hypoglycemic states.  Pilot work (Fig. 4) shows a standard deviation of the 
control group minus patient group difference during the hypoglycemic state of 0.25 µmol/g, indicating that with 
10 participants we have 85% power to detect paired-group differences of 0.27 µmol/g with type I error of 5%. 
In the case that standard deviations are twice those observed in Figure 4, we have 85% power to detect 
differences of 0.53 µmol/g. Preconditioning: Similar comparisons will be carried out between GABA levels 
during control euglycemia states for euglycemic preconditioning compared to hypoglycemic preconditioning. 
This will be repeated for GABA during hypoglycemic states. 
Potential pitfalls and solutions: 
Prior rodent studies that demonstrated a role for increased hypothalamic GABA tone in blunting the 
counterregulatory response to hypoglycemia studied the VMH, while our spectroscopic voxel contains a larger 
portion of the hypothalamus.  Despite this pitfall, our preliminary data demonstrate our ability to measure the 
hypothalamic GABA response to hypoglycemia both in controls and patients with type 1 diabetes.  Therefore, 
we expect to be able to address our hypotheses regarding hypothalamic GABA despite these partial volume 
effects.   
 As discussed above, one potential problem we may encounter is that the protocol we use to induce HAAF 
will not be effective for all subjects enrolled in the study.  To ensure that HAAF is present at the time we 
examine GABA metabolism, we will measure counterregulatory hormones in blood collected from subjects 
during hypoglycemia.  If the epinephrine response (the hormone most blunted during most protocols in which 
HAAF is induced in humans) during the study done after hypoglycemic preconditioning is > 70% of the 
response measured during the study after euglycemic preconditioning, we will conclude that HAAF was not 
induced and the data will not be used for analysis.  If HAAF fails to be induced in 1/3, 2/5, 3/7, 4/9,... of the 
studies (we will monitor in an ongoing way), we will change the protocol to induce HAAF to include 
more episodes of hypoglycemia before the GABA experiment.  In addition, in this aim the blunted 
counterregulation will also be confirmed in patients with type 1 diabetes by comparing their 
hormone measurements during the hypoglycemic clamp to those of the euglycemia-preconditioned 
controls. 
Research Strategy
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Principal Investigator/Program Director (Last, first, middle): Seaquist, Elizabeth, R.

ICF_000.pdf: 
IRB approval date: April 8, 2014 
9412M09040 
page 1 of 8 
CONSENT FORM 
(Revised April 8, 2014) 
Measurement of Glucose/Glycogen Metabolism in Humans Using 
Magnetic Resonance at 4 or 7 Tesla 
You are invited to participate in a protocol designed to investigate the effects of diabetes mellitus and 
high blood insulin on the brain, heart and skeletal muscle using magnetic resonance.  Some participants 
will also be asked to be part of a teaching program designed to prepare people for protocols done in a 
magnetic resonance scanner.  You were selected as a possible participant because you are healthy, are 
not pregnant and you have no known medical disease and therefore your glucose metabolism will be 
typical of a normal person.  Alternatively, you may have been asked to participate because you have 
diabetes. 
We ask that you read this form and ask any questions you may have before agreeing to be in the 
protocol.  This protocol is being conducted by: 
 
 
Elizabeth R. Seaquist, M.D. 
Gulin Oz, Ph.D. 
Department of Medicine 
Department of Radiology 
(612) 626-4833 
(612) 625-7897 
University of Minnesota Medical School 
Minneapolis, MN  55455 
Background Information: 
The blood sugar called glucose is a major fuel for most organs in the human body, particularly the brain.  
How and where the body uses glucose is regulated by a number of hormones, for instance insulin and 
glucagon, which regulate how glucose is stored in tissues as glycogen.  In a number of diseases, in 
particular diabetes mellitus, the glucose supply to the brain may be different than in normal subjects and 
brain glycogen metabolism may be deranged, which may be a cause for reduced awareness of low blood 
sugar. 
The purpose of this protocol is to determine the effects of altered glucose metabolism on the brain.  For 
example, patients with long duration diabetes mellitus lose their ability to secrete the hormones 
necessary to protect them against hypoglycemia (low blood sugar), which may be due to alterations in 
glucose availability to the human brain. 
To measure these effects, we will use intravenous infusions of glucose and insulin (both of which occur 
naturally and are made by your body) and a magnetic resonance 4 Tesla or 7 Tesla scanner. 
How the Protocol is Performed: 
If you agree to participate, you will be asked to come to the Center for Magnetic Resonance Research 
(CMRR) for a tour of the facility in the days before the actual experiment.  While at the CMRR, one of 
the investigators will talk with you about the protocol and provide you with information about magnetic 
resonance scanning. 
Before participating in the protocol we will ask you not to use cigarettes, alcohol, or caffeine for at least 
8 hours before the start time of the visit. In addition you will be asked not to engage in heavy exercise or 
strenuous physical activity 24 hours prior to testing. All subjects will be asked to fast for a minimum of 
8 hours before arriving at the center on Days 1 and 2.  Healthy volunteers will be asked to participate in 
the protocol up to 5 days, and volunteers with diabetes will be asked to participate up to 4 days. 


IRB approval date: April 8, 2014 
9412M09040 
page 2 of 8 
Healthy controls 
If you are a healthy control you will first be asked to participate in 3 experiments involving either 
episodes of or euglycemia (normal blood glucose – Protocol A in the Figure) hypoglycemia (low blood 
glucose – Protocol B in the Figure). These experiments will be done over 2 days. You will be asked to 
participate in both protocols A and B as illustrated in the Figure The protocols will be separated by 
minimum of 6 weeks. From the day before each protocol starts until the conclusion of each protocol, we 
will ask you to avoid strenuous exercise. On the days 1 and 2 you will be asked to not eat anything after 
midnight. Both protocols will be done at the Masonic Clinical Research Center (MCRU) and CMRR. 
 
 
 


IRB approval date: April 8, 2014 
9412M09040 
page 3 of 8 
On day 1 for each protocol you will arrive at the MCRU at 7 AM. On Day 1 in the Protocol A you will 
undergo two 120 minutes experiments in which blood glucose will be maintained in the normal range. In 
Protocol B, there will be two 120 minutes experiments of hypoglycemia on day 1. To prepare for each 
protocol, a small intravenous catheter will be placed in your arm for infusion of insulin, potassium and 
glucose. Insulin is a hormone that your body makes naturally and it causes your blood sugar to decrease. 
Glucose is a sugar and it is used to carefully regulate your blood sugar level and to ensure that your 
blood sugar level does not go too low. Potassium is a salt-like substance that is present in the blood. A 
second catheter will be placed in your other arm for venous blood sampling. This arm will be wrapped in 
a heating pad to ensure adequate blood flow and accurate laboratory results. Blood will be drawn every 5 
minutes for measurement of glucose and other hormones. Insulin and glucose will be infused in 
calculated doses to maintain your blood sugar level in the target range. At the end of the 
euglycemic/hypoglycemic experiment all infusions will be discontinued and the blood sugar level 
allowed to normalize. At this time you will be given a meal and when the blood glucose returns to 
normal you will be allowed to go home. 
On day 2, you will again arrive at the MCRU at 7 AM and the same protocol will be followed as in the 
morning on day 1. On day 2 there will be one 120 minute experiment of euglycemia in protocol A and 
one 120 minute experiment of hypoglycemia in protocol B. After arriving, a small intravenous catheter 
will be placed in your arm for infusion of insulin, potassium and glucose. A second catheter will be 
placed in your other arm for venous blood sampling. This arm will be wrapped in heating pad to ensure 
adequate blood flow and accurate laboratory results. Blood will be drawn every 5 minutes for 
measurement of glucose and other hormones. Insulin and glucose will be infused in calculated doses to 
maintain your blood sugar level in the target range. At the end of the euglycemic/hypoglycemic 
experiment all infusions will be discontinued and the blood sugar level allowed to normalize. You will 
then be given a small dose of glucose enriched with 13C label via the catheter.  This infusion will 
continue until the evening of day 5 and is necessary for us to measure how your brain is using glucose.  
Small plastic catheters will remain in two veins in your arms for infusion of glucose and for measuring 
your blood glucose levels every 15-120 minutes.  During the protocol we will also ask you to give a 
urine sample every 1-4 hours.  The urine sample and some of the blood samples will be saved for studies 
of metabolism without any information that identifies the samples as yours. You will spend most of your 
time in a hospital bed, but will be allowed to use the restroom and walk around briefly.  Meals will be 
provided throughout each day as shown in the Figure, but will be very low carbohydrate in order to 
minimize your own insulin release. Snacks without carbohydrate will also be available.  IV catheters will 
be kept in place from the start of day 2 until the end of the protocol on day 5. 
Starting on day 2 in each protocol, you will be asked to transfer to the Center for Magnetic Resonance 
Research twice a day.  On day 2 the transfer will be in the afternoon and the evening.  On days 3, 4, and 
5 transfer will be in the mornings and evenings.  At the CMRR you will be asked each time to conduct 
an MRI scan as follows: You will be placed on the patient bed in the magnet room for one-to-two hours. 
A nurse or researcher will stay with you at all times during this protocol.  When the scan is completed 
you will be transferred back to your hospital room in the MCRU. After the final scan on day 5, the 
infusions will be stopped, the catheters will be removed, and you will be fed a normal meal. 
You may also be asked several days prior to the protocol to report to the Clinical Research Center for a 
"mock" protocol to see if you are comfortable with the MRI scan and/or to test your body’s response to 
either protocol (as described above, but carried out at the Clinical Research Center). If you would like to 
try the MRI procedure in advance, this test will not exceed 2 hours. If we need to test your body's 
response to the infusion, this “pre-test” will involve only drawing blood and administering glucose and 
will be performed as described for either protocol above, but will last not more than 4 hours. 
 


IRB approval date: April 8, 2014 
9412M09040 
page 4 of 8 
Magnetic resonance measurements performed in the magnet are very similar to magnetic resonance 
imaging exams used for diagnosis.   The researcher will be watching you at all times.  You will be given 
a bulb to hold during the measurements, by squeezing the bulb, you can signal the researcher if you want 
to stop.  The magnetic resonance measurements will be associated with regular loud clunking noises that 
you may find unpleasant.  For your comfort, you will be given ear protection to wear while lying in the 
scanner. 
The MRI scan being done is designed to answer research questions, not for diagnosis of a medical 
condition.  The research MRI scan is not a substitute for one a doctor would order for diagnostic 
imaging.  It may not show problems that would be picked up by a clinical MRI scan.  However, if we 
believe that we may have found a medical problem in your MRI scan, we will ask a doctor who is 
trained in the reading of MRI scans (a radiologist) to help us review the scan.  If the radiologist thinks 
there may be an abnormality in your MRI scan, we will contact you and, with your permission, help you 
contact a doctor to obtain medical follow-up. 
The researchers will give you a questionnaire to complete after the protocol.  This should take no more 
than 5 minutes to complete. 
Participants with diabetes 
Participants with diabetes will start the protocol in the MCRU on the afternoon of Day 2 and only 
participate in one 5 day protocol.  Participants with diabetes will not undergo the 
euglycemic/hypoglycemic experiment on days 1 and 2, but will only do the MRI scans on days 2-5, The 
researcher will advise you on how to adjust your insulin regimen during the 1-2 days before the protocol 
starts so your blood sugar is in good control when you arrive at the MCRU.  After arrival, an IV will be 
placed in each of your arms. This will allow for giving insulin, glucose and potassium during the test. 
This catheter will also allow us to collect blood during the test.  You will then be given a small dose of 
glucose enriched with 13C label via the catheter.  This infusion will continue until after the last MRI scan 
on day 5 and is necessary for us to measure how your brain is using glucose.  Small plastic catheters will 
remain in two veins in your arms for infusion of glucose and for measuring your blood glucose levels 
every 15-120 minutes.  During the protocol we will also ask you to give a urine sample every 1-4 hours.  
The urine sample and some of the blood samples will be saved for studies of metabolism without any 
information that identifies the samples as yours. You will spend most of your time in a hospital bed, but 
will be allowed to use the restroom and walk around briefly.  Meals will be provided throughout each 
day as shown in the Figure, but will be very low carbohydrate. Snacks without carbohydrate will also be 
available.  IV catheters will be kept in place from the start of day 2 until the end of the protocol on day 5. 
Starting on day 2 in each protocol, you will be asked to transfer to the Center for Magnetic Resonance 
Research twice a day.  On day 2 the transfer will be in the afternoon and the evening.  On days 3, 4, and 
5 transfer will be in the mornings and evenings.  At the CMRR you will be asked each time to conduct 
an MRI scan as follows: You will be placed on the patient bed in the magnet room for one-to-two hours. 
A nurse or researcher will stay with you at all times during this protocol.  When the scan is completed 
you will be transferred back to your hospital room in the MCRU. After the final scan on day 5, the 
infusions will be stopped, the catheters will be removed, and you will be fed a normal meal. 
You may also be asked several days prior to the protocol to report to the Clinical Research Center for a 
"mock" protocol to see if you are comfortable with the MRI scan and/or to test your body’s response to 
either protocol (as described above, but carried out at the Clinical Research Center). If you would like to 
try the MRI procedure in advance, this test will not exceed 2 hours. If we need to test your body's 
response to the infusion, this “pre-test” will involve only drawing blood and administering glucose and 
will be performed as described for either protocol above, but will last not more than 4 hours. 
 


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Magnetic resonance measurements performed in the magnet are very similar to magnetic resonance 
imaging exams used for diagnosis.   The researcher will be watching you at all times.  You will be given 
a bulb to hold during the measurements, by squeezing the bulb, you can signal the researcher if you want 
to stop.  The magnetic resonance measurements will be associated with regular loud clunking noises that 
you may find unpleasant.  For your comfort, you will be given ear protection to wear while lying in the 
scanner. 
The MRI scan being done is designed to answer research questions, not for diagnosis of a medical 
condition.  The research MRI scan is not a substitute for one a doctor would order for diagnostic 
imaging.  It may not show problems that would be picked up by a clinical MRI scan.  However, if we 
believe that we may have found a medical problem in your MRI scan, we will ask a doctor who is 
trained in the reading of MRI scans (a radiologist) to help us review the scan.  If the radiologist thinks 
there may be an abnormality in your MRI scan, we will contact you and, with your permission, help you 
contact a doctor to obtain medical follow-up. 
The researchers will give you a questionnaire to complete after the protocol.  This should take no more 
than 5 minutes to complete. 
Potential Risks and Benefits: 
The protocol has several risks: 
1. 
Magnetic field:  Magnetic resonance examinations have become a routine tool in today’s medical 
diagnosis.  Based on more than a decade of experience with these devices, appropriate guidelines exist 
for these exams: 
We have taken measures to eliminate the potential risk of the danger of magnetic objects being attracted 
by the magnet.  We have elaborate procedures to make sure that no one brings anything magnetic into 
the room.  Another potential risk is presented by the radio waves used. However, these will be applied 
according to guidelines set forth by the Food and Drug Administration (FDA).  The magnet we will use 
has a static field strength that is several times that used for clinical exams.  There is a risk of unknown 
effects related to participation in 4 or 7 Tesla MRI research.  Long term effects of exposure to high 
magnetic fields are unknown.  Short term most people experience no ill effects from the strong magnetic 
field, but some people report dizziness, mild nausea, headache, a metallic taste in their mouth, or 
sensations of flashing lights. These symptoms, if present, subside shortly after leaving the magnet. If any 
sensations experienced during participation cause discomfort or pain, notify the researcher right away 
and your participation will stop and you will be taken out of the magnetic field. 
2. 
Infusions/blood drawing:  Occasionally individuals may have some bruises afterwards where the 
catheter was inserted.  Rarely an individual may experience inflammation of the vein (phlebitis) but this 
usually goes away with local heat and elevation of the extremity. We would not wish to you to 
participate in the protocol if you have given or expect to give a similar amount of blood in the month 
before or after the clamp.  We will draw at most 7 oz. of blood (approximately 200 ml). Infusion of 
insulin (a hormone made naturally by the human body) produces no side effects other than the potential 
risk of low blood sugar, which we will avoid as described below. 
3. 
Hypoglycemia (low blood sugar): Symptoms of hypoglycemia may occur when your blood sugar 
falls below 60 mg/dl and include sweating, shakiness, confusion, increased heartbeat and feeling “low”.  
It is reversed within minutes by stopping the insulin infusion and by raising your body sugar by 
increasing the glucose infusion.  If the blood sugar drops very low (lower than the level at which we will 
perform the protocol), seizures or abnormalities in heart rhythm can occur.  To be certain that your blood 
sugar level does not drop too low, we will check your blood every five minutes during insulin infusion 


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and adjust the amount of sugar we give you by vein to keep your blood sugar at the correct level.  With 
such careful monitoring, it is very unlikely that you will develop a blood sugar low enough to make you 
feel ill.  Your blood sugar will be regulated to be in the normal range or higher during the magnet 
protocol. 
4. 13C glucose:  The stable isotope 13C is naturally present in your body and 13C glucose in its pure form 
has no known side effects different from that of regular glucose.  This isotope is not radioactive.  We 
have carefully established procedures to minimize the risk of impurities in the glucose infused. We will 
infuse glucose enriched with this isotope, which we will then measure using the magnet. 
5. Urine collection:  Some people find it difficult to provide a urine sample into a collection vessel.  To 
make this as easy as possible for you, you will be given privacy during the collection. 
Potential Benefits: 
There are no direct benefits of participation to you other than the satisfaction you may have in helping us 
answer important as yet unanswered questions about human glucose metabolism. 
Alternatives to Participating in the Protocol: 
You do not have to participate in this protocol. 
Research Related Injury: 
In the event this research activity results in injury, treatment will be available, including first aid, 
emergency treatment and follow-up care as needed. Care for such injuries will be billed in the ordinary 
manner, to you or your insurance company. If you think that you have suffered a research related injury 
let the protocol physicians know right away. 
Protocol Costs/Compensation: 
You will be paid $250 to help cover travel expenses and time lost from work for each day of the 
protocol. Subjects participating in a series of experiments over more than one day will be paid an 
additional $50 at the completion of the protocol. If you would like to test your comfort level with the 
MRI scan several days prior to the protocol, you will be reimbursed separately for this scan at a rate of 
$15/hour spent for the whole procedure. 
Confidentiality: 
The records of this protocol will be kept private.  In any sort of report we might publish, we will not 
include any information that will make it possible to identify a subject.  Your record for the protocol 
may, however, be reviewed by representatives of the Food and Drug Administration and by departments 
at the University with appropriate regulatory oversight.  To that extent, confidentiality isn’t absolute. 
Protocol data will be encrypted according to current University policy for protection of confidentiality. 
Voluntary Nature of the protocol: 
Participation in this protocol is voluntary. Your decision whether or not to participate will not affect your 
current or future relations with the University or the CMRR.  If you decide to participate, you are free to 
withdraw at any time without affecting those relationships. 
New Information: 
If during the course of this research protocol there are significant new findings discovered which might 
influence your willingness to continue, the researchers will inform you of those developments. 
 


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Notification of Significant New Findings: 
Personnel in the Center for Magnetic Resonance Research (CMRR), the site where you are participating 
in MRI research, maintain a list of the names and contact information of all participants included in 
research at this facility.  This information is required and will be used by CMRR to notify participants of 
significant new information about the effects of MR on human health that develop over the course of 
MRI research.  Participant’s identifying information is stored securely and it is maintained in a 
confidential manner by persons with oversight of research conducted at the CMRR. 
 
Incidental Findings: 
The images or pictures created during this protocol are for research purposes only and are not intended to 
provide health care to you. However, if the results from the magnetic resonance imaging show 
something unusual in the pictures, a Radiologist trained in reading the pictures will look at them. The 
pictures will not contain any personal information except your age and pertinent medical history 
collected as part of the research. There will be no charge to you for having the Radiologist look at your 
pictures. The investigator will contact you if the recommendation of the Radiologist is to further 
investigate the unusual results of the pictures with your own physician. However, further medical follow 
up is not a part of this protocol and the protocol does not have funds set aside for this purpose. 
Therefore, if the results do show something unusual, any medical follow up cost will be your 
responsibility and/or the responsibility of your health insurance carrier. 
Contacts and Questions: 
The researchers conducting this protocol are Drs. Elizabeth R. Seaquist and Gulin Oz.  You may ask any 
questions you have now.  If you have any questions later, you are encouraged to contact them at (612) 
626-4833 (E.R.S.),  or (612) 625-7897 (G.O.) 
If you have any questions or concerns regarding the protocol and would like to talk to someone other 
than the researchers, you are encouraged to contact Research Subjects’ Advocate Line, D528 Mayo, 
420 Delaware St. Southeast, Minneapolis, Minnesota 55455; (612) 625-1650. 
Statement of Consent: 
I have read the above information.  I have asked questions and have received answers.  I consent to 
participate in the protocol. 
You will be given a copy of this form to keep for your records. 
 
 
Signature:   
 
 Date:  
 
Signature of Investigator:  
 Date:  
 
 
 


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Statement of Consent for storage of blood and urine samples 
I agree that the investigators may store samples of my blood and urine for the protocol of metabolism 
and understand that they will be stored without any information that would identify the samples as mine. 
 
Signature:   
 
 Date:  
 
 
I do not want the investigators to store my samples. 
 
Signature:   
 
 Date: