Impact of Recurrent Hypoglycemia on Brain Ketone Metabolism in Type 1 Diabetes
Phase: PHASE1
Conditions: Type 1 Diabetes, Hypoglycemia
Sponsor: Yale University

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SECTION I: ADMINISTRATIVE INFORMATION
YALE UNIVERSITY
HUMAN INVESTIGATION COMMITTEE
Application to Involve Human Subjects in Biomedical Research 
100 FR1 (2011-3)
HIC OFFICE USE ONLY
Please refer to the HIC website for application 
instructions and information required to 
complete this application. The Instructions are 
available at http://www.yale.edu/hrpp/forms- 
templates/biomedical.html
Submit the original application and two (2) 
copies of all materials including relevant 
sections of the grant which funds this project 
(if applicable) to the HIC.
DATE STAMPED-RECEIVED
PROTOCOL NUMBER
Title of Research Project: Impact of Hypoglycemia on Brain Ketone and Neurotransmitter Metabolism in 
Type 1 DM.
Principal Investigator:
Raimund Herzog, MD
Yale Academic Appointment:
Associate Professor of Internal Medicine
Campus Address:
300 Cedar Street, TAC S131
Campus Phone: 737-4773
Fax: 737-5558
Pager:
E-mail: raimund.herzog@yale.edu
Protocol Correspondent Name & Address (if different than PI):
Jessica Leventhal, APRN
Campus Phone: 475.331.8966
Fax:
E-mail: jessica.leventhal@yale.edu
Yale Cancer Center CTO Protocol Correspondent Name & Address (if applicable): 
N/A
Campus Phone:
Fax:
E-mail:
Faculty Advisor:(required if PI is a student, 
resident, fellow or other trainee)
 NA
Yale Academic Appointment:
Campus Address:
Campus Phone:
Fax:
Pager:
E-mail:
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SECTION II: GENERAL INFORMATION
Investigator Interests:
Does the principal investigator, co-investigator, or any other responsible research team member, or any of their family 
members (spouse, child, domestic partner) have an incentive or interest, financial or otherwise, that may be viewed as 
affecting the protection of the human subjects involved in this project, the scientific objectivity of the research or its 
integrity? See Disclosures and Management of Personal Interests in Human Research 
http://www.yale.edu/hrpp/policies/index.html#COI
 Yes
X No
If yes, list names of the investigator or responsible person:
The Yale University Principal Investigator and all Yale University and Yale New Haven Hospital individuals who 
are listed as co-investigators on a protocol with a Yale University Principal Investigator must have a current 
financial disclosure form on file with the University’s Conflict of Interest Office. If this has not been done, the 
individual(s) should follow this link to the COI Office Website to complete the form: http://www.yale.edu/coi/
NOTE: The requirement for maintaining a current disclosure form on file with the University’s Conflict of Interest 
Office extends primarily to Yale University and Yale-New Haven Hospital personnel. Whether or not they are 
required to maintain a disclosure form with the University’s Conflict of Interest Office, all investigators and 
individuals deemed otherwise responsible by the PI who are listed on the protocol are required to disclose to 
the PI any interests that are specific to this protocol.
1.
Performing Organizations: Identify the hospital, in-patient or outpatient facility, school or other agency that 
will serve as the location of the research. Choose all that apply:
a. Internal Location[s] of the Study:
 Magnetic Resonance Research Center
PET Center
(MR-TAC)
YCCI/Church Street Research Unit (CSRU) 
Yale Cancer Center/Clinical Trials Office (CTO)
YCCI/Hospital Research Unit (HRU)
Yale Cancer Center
YCCI/Keck Laboratories
Yale-New Haven Hospital
Cancer Data Repository/Tumor Registry 
Specify Other Yale Location:
b. External Location[s]:
APT Foundation, Inc.
Haskins Laboratories 
Connecticut Mental Health Center
John B. Pierce Laboratory, Inc.
Clinical Neuroscience Research Unit(CNRU)
Veterans Affairs Hospital, West Haven 
Other Locations, Specify:
International Research Site
(Specify location(s)):
c. Additional Required Documents (check all that apply):
N/A
*YCCI-Scientific and Safety Committee (YCCI-SSC)
Approval Date:
*Pediatric Protocol Review Committee (PPRC)
Approval Date:
*YCC Protocol Review Committee (YRC-PRC)
Approval Date:
*Dept. of Veterans Affairs, West Haven VA HSS
Approval Date:
*Radioactive Drug Research Committee (RDRC)
Approval Date: 
YNHH-Radiation Safety Committee (YNHH-RSC)
Approval Date:
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 Magnetic Resonance Research Center PRC (MRRC-PRC) Approval Date: June 15, 
2012
YSM/YNHH Cancer Data Repository (CaDR)
Approval Date: 
Dept. of Lab Medicine request for services or specimens form
*Approval from these committees is required before final HIC approval is granted. See 
instructions for documents required for initial submission and approval of the protocol. 
Allow sufficient time for these requests. Check with the oversight body for their time 
requirements.
2. Probable Duration of Project: State the expected duration of the project, including all 
follow-up and data analysis activities. 10 years
3. Research Type/Phase: (Check all that apply)
a. Study Type
Single Center Study 
Multi-Center Study
Does the Yale PI serve as the PI of the multi-site study? Yes
No 
Coordinating Center/Data Management
Other:
b. Study Phase
N/A
Pilot
Phase I
Phase II
Phase III
Phase IV 
Other (Specify)
4. Area of Research: (Check all that apply) Note that these are overlapping definitions and 
more than one category may apply to your research protocol. Definitions for the following 
can be found in the instructions section 4c:
Clinical Research: Patient-Oriented
Clinical Research: Outcomes and 
Clinical Research: Epidemiologic and Behavioral
Health Services 
Translational Research #1 (“Bench-to-Bedside”)
Interdisciplinary Research 
Translational Research #2 (“Bedside-to-Community”)
Community-Based
Research
5. Is this study required to be registered in a public database? Yes
No 
 
If yes, where is it registered?
Clinical Trials.gov registry 
Other (Specify)
6. Will this study have a billable service as defined by the Billable Service Definition? 
Yes
No
If you answered "yes", this study will need to be set up in Patient Protocol Manager (PPM)
http://medicine.yale.edu/ymg/systems/ppm/index.aspx
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TY IRB 8/19/2020
Figure 1: Model of cerebral metabolism in 
neuronal and astroglial compartments and 
metabolite 13C labeling patterns following 
the 
intravenous 
infusion 
of 
[1-13C]- 
glucose (black circle), [2-13C]-glucose 
(gray circle) or [2-13C]-acetate (black 
circle). Glucose enters both neuronal and 
astroglial 
compartments 
and 
labels 
pyruvate and subsequently lactate through
SECTION V: RESEARCH PLAN
7. Are there any procedures involved in this protocol that will be performed at YNHH or one of 
its affiliated entities? Yes 
No _X 
If Yes, please answer questions a through c and 
note instructions below. If No, proceed to Section III.
a.
Does your YNHH privilege delineation currently include the specific procedure that 
you will perform?
n/a
b. Will you be using any new equipment or equipment that you have not used in the past 
for this procedure?
n/a
c.
Will a novel approach using existing equipment be applied?
n/a
If you answered “no” to question 7a, or "yes" to question 7b or c, please contact the YNHH 
Department of Physician Services (688-2615) for prior approval before commencing with your 
research protocol.
1.
Statement of Purpose: State the scientific aim(s) of the study, or the hypotheses to be tested.
To determine the long-term effect of severe antecedent hypoglycemia in T1DM patients 
on brain energy substrate metabolism, in particular alternate energy substrate and 
glucose metabolism. We will measure brain fuel uptake kinetics across the blood brain 
barrier into the brain, brain glucose levels and neuronal and glial TCA cycle activity. We 
will further explore the relationships between absolute neurotransmitter and brain 
metabolite levels in healthy control subjects and Type 1 diabetes subjects and how they 
vary across different brain regions.
2.
Background: Describe the background information that led to 
the plan for this project. Provide references to support the 
expectation of obtaining useful scientific data.
2.1.1. Counterregulatory failure in intensely treated 
diabetic patients.
The long-term impact of hypoglycemia on brain 
function and development is becoming clearer {Ho, 
2008 #1731} and confirms the long held fear of 
hypoglycemia as the most dreaded immediate 
complication of T1DM treatment with insulin {DCCT, 
1997 #1361;Flykanaka-Gantenbein, 2004
4
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#1391;Kodl, 2008 #1693}. Complications of uncontrolled hyperglycemia in diabetic 
patients are well established and can be avoided via intensive insulin therapy {DCCT, 
1993 #1245;UKPDS, 1998 #1360}. Constrained by these two extremes, measures that 
allow for intensive glucose control without a negative impact on normal brain function are 
still needed. The counterregulatory mechanisms to low glucose include responses on 
several levels: In the pancreas insulin secretion is suppressed; glucagon and epinephrine 
are increased and conscious measures are taken to reverse this possibly life-threatening 
state {Cryer, 1994 #1238}. One or several of these are compromised in T1DM patients 
when compared to non-diabetic individuals mostly by affecting the responsible centers in 
the brain in their control {Amiel, 1987 #1363;Amiel, 1988 #1364}. The experiments 
proposed here are part of the effort to identify ways that would protect the brain 
from serious hypoglycemic injury.
2.1.2. Characterization of brain metabolism in vivo by NMR spectroscopy.
In vivo biochemical assessment of metabolism by NMR spectroscopy using energy 
substrates labeled with the stable carbon isotope 13C/2H allows real time observation of 
neurotransmitter cycle activity in the brain (11; 12). Glutamine-glutamate neurotransmitter 
cycling is stochiometrically coupled to the turns of the TCA cycle, thereby providing the 
basis for the calculation of TCA cycle flux in astrocytes and neurons in a model of brain 
metabolism (1)
(Figure 1). This method allows characterization of fuel uptake across the blood brain 
barrier as well as brain metabolism under different physiological and disease conditions. 
Infused acetate is predominantly metabolized in glial cells, while labeled lactate, beta- 
hydroxy-butyrate (BHB) or glucose are preferably metabolized in neurons, where lactate 
and glucose (but not BHB) enter the TCA cycle by conversion of pyruvate to acetyl-CoA, 
a reaction catalyzed by the pyruvate dehydrogenase complex (PDH) (13-16). The effects 
of peripheral changes on plasma metabolite concentrations are very closely monitored 
and accounted for by frequent plasma sampling and incorporation into the metabolic 
model (17). The recent observation by members of our group that infused acetate showed 
enhanced uptake and increased metabolic flux in the cortex of patients with longstanding, 
but well controlled T1DM (18) led us to perform similar experiments in our animal model 
of three days of recurrent hypoglycemia (3dRH). Application of NMR spectroscopy to 
the study of brain fuel metabolism in our in vivo animal model of recurrent 
hypoglycemia allowed us to measure brain TCA cycle activity in real time. This is 
particularly relevant to the metabolic adaptations in T1DM patients.
2.1.3. In vivo measurement of brain acetate and lactate metabolism and determination 
of TCA cycle activity.
From the infusion of labeled 13C-acetate into control and recurrently hypoglycemic 
animals under eu- and hypoglycemia we gained valuable new insights that are now 
guiding our choice of alternate fuels: Our experiments recapitulated the increase in fuel 
uptake observed in the human study conducted by our group (18). Due to the ability to 
infuse larger doses of acetate that allowed us to achieve higher blood concentrations than 
is ethically permissible in humans, we were able to determine that fluxes contributing to 
neuronal metabolism were particularly affected by antecedent recurrent hypoglycemia
5
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(Figure 2). At euglycemia 
we observed a striking 
increase in basal glucose 
consumption 
(CMRglc) 
following 
pretreatment 
with 
three 
bouts 
of 
hypoglycemia 
in 
the 
3dRH 
animals 
when 
compared 
to 
controls, 
with only a small change 
in 
acetate 
utilization 
(CMRac). 
In 
contrast 
under 
hypoglycemia, 
whole 
brain 
glucose 
utilization
markedly 
dropped in the 3dRH 
group, while no such 
change was observed in 
controls.
We were able to attribute 
this net change to a 
neuronal 
compartment- 
specific 
response 
to 
hypoglycemia: 
Animals 
from 
both 
groups 
decreased their glucose
Figure 2: a) Calculated flux rates for key metabolic pathways in the neuronal and 
astrocytic compartment. (controls euglycemia - black, controls hypoglycemic – grey, 
3dRH euglycemia – white, 3dRH hypoglycemia – hatched) b) Percent change in flux 
between euglycemia and hypoglycemia. (controls – black, 3dRH – white). CMRac= 
acetate oxidation rate; CMRgl= glucose oxidation rate; Vcyc= glutamine-glutamate cycle 
flux; Vgln= glutamine synthase flux; VpdhA= astrocytic PDH flux; VpdhN= neuronal PDH 
flux; VtcaA= astrocytic TCA cycle flux; VtcaN= neuronal TCA cycle flux.
and lactate utilization in astrocytes under hypoglycemia, whereas neuronal pyruvate 
dehydrogenase flux (VpdhN) increased in control animals, but markedly decreased in 3dRH 
animals by 50%. This difference translates further into parallel changes of the neuronal 
TCA cycle rates (VtcaN) between groups (Figure 2), suggesting a subsequent decrease in 
ATP production and energy deficit. Thus while control animals can increase neuronal TCA 
cycle activity during hypoglycemia via enhanced
substrate influx through pyruvate dehydrogenase, 
animals pre-exposed to recurrent hypoglycemia do 
not show such compensation.
These findings suggest that a more neuron-specific 
alternate fuel would be required to prevent neuronal 
energy deficits under acute hypoglycemia in the 
clinical setting, since acetate is predominantly 
supporting astroglial metabolism. Such a candidate 
fuel that can be safely delivered to humans is the 
ketone body BHB. It is, like acetate, taken up into the 
brain via monocarboxylic acid transporters (19). This 
makes it a particularly attractive candidate molecule, 
since its brain levels would be enhanced under the
very  circumstances  under  which  it  is supposed to
Figure 3: Time courses of 13C label
appearance in cortex and subcortex of
control and 3dRH pretreated animals; 
n=5.  Single  exponential  fits.  (glu4=
6
glutamate  carbon  position  4;  gln4=
arbon  position  4;  fE%=
fractional enrichment in %).
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support metabolism. Exciting results from a study of infused lactate -another 
predominantly neuronal fuel - in control and 3dRH animals at euglycemia have revealed 
a faster label appearance in the metabolites glutamine and glutamate (Figure 3), 
suggesting enhanced neuronal lactate metabolism after recurrent hypoglycemia and 
confirming our initial hypothesis.
We have identified changes in fuel transport into the brain and further utilization 
via neuronal PDH that may underlie the adaptations seen in T1DM patients. BHB 
may be particularly well suited to compensate for this deficit since it is taken up by 
the transporters known to be upregulated after exposure to antecedent recurrent 
hypoglycemia and it particularly supports the neuronal compartment.
2.1.4. Altered Brain Neurotransmitter Homeostasis in Type 1 Diabetes
Following a hypoglycemic period of 2 hours and
an infusion of lactate in our animal model, we 
have observed significant changes in brain 
metabolite concentrations, when compared to 
euglycemic controls (Figure 4). Particularly the 
neurotransmitters aspartate, glutamate and 
GABA were altered, suggesting changes in the 
neural excitability of the volume of brain cortex 
we studied. Whether such a dramatic change in 
neurotransmitter homeostasis accompanies the 
physiologic response to hypoglycemia and how 
it is affected by recurrent hypoglycemia in
300
250
200
150
100
50
0
-50
-100
Asp
Gln
Glu
GABA
ala
lac
humans and is not known at this point and will 
be studied under this proposal. Proton NMR 
measurements at high resolution at 7Tesla 
will allow us to characterize any progressive 
metabolic disturbance in neurotransmitter
Figure 4: Percent change of brain
neurotransmitter levels in control (white 
bars) and recurrently hypoglycemic (filled 
bars), comparing clamped euglycemia with 
clamped hypoglycemia.
level that may occur in the context of recurrent hypoglycemia.
2.1.5. Effect of Ketone Supplementation on Cognition under Hypoglycemia.
Cognitive impairment is the most serious adverse consequence of insulin-induced 
hypoglycemia. It is unknown to what degree hypoglycemia-induced cognitive dysfunction 
can be alleviated in T1DM subjects by peripheral administration of BHB as an alternate 
energy source for the brain. In contrast to lactate, our MR spectroscopy measurements 
of brain metabolism in humans suggest that intensively treated T1DM patients with 
hypoglycemia unawareness could particularly benefit from oral ketone (e.g. ester) 
supplementation, which already is safely being used in neurodegenerative diseases many 
of which are now known to have a neuro-metabolic basis, such as Alzheimer’s disease. 
For this purpose, we will conduct studies designed to examine the potential value of this 
approach by infusing BHB (or placebo) in T1DM subjects during a hyperinsulinemic- 
hypoglycemic clamp in conjunction with testing of cognitive function. This will lay the basis 
for subsequent trials exploring the novel use of therapeutic doses of oral ketone
ctrl_hypoclamp-euclamp 
Rhhypoclamp-euclamp
Conc [mM]
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formulations (such as ketone esters) that are currently under commercial development, 
which could serve as adjunct therapies to protect the brain from hypoglycemia in T1DM.
2.1.6. Influence of lactate on brain 13C/2H-glucose metabolism under hypoglycemia in 
intensively treated T1DM subjects with hypoglycemia unawareness
We have made the surprising observation in our animal model that infused lactate does 
not contribute to the increased oxidation rate of RH animals during hypoglycemia [20], 
which has since been supported by studies in T1DM patients [21]. In further follow-up 
experiments, we used DCA, an inhibitor of brain pyruvate dehydrogenase kinase isoforms 
and modulator of glucose metabolism, to demonstrate a possible important role for 
regulation of pyruvate dehydrogenase flux under hypoglycemia in an animal model of RH 
that recapitulated the effects of lactate. No study to date has directly examined the 
interaction of glucose and lactate metabolism under hypoglycemia in the brain of 
intensively treated T1DM subjects. Novel studies conducted under this protocol will yield 
this critical new information needed to tailor our therapeutic approaches to protect the 
brain from hypoglycemic injury.
3.
References
1. de Graaf RA, Mason GF, Patel AB, Behar KL, Rothman DL: In vivo 1H-[13C]-NMR 
spectroscopy of cerebral metabolism. NMR Biomed 16:339-357, 2003
2. Ho MS, Weller NJ, Ives FJ, Carne CL, Murray K, Vanden Driesen RI, Nguyen TP, Robins PD, 
Bulsara M, Davis EA, Jones TW: Prevalence of structural central nervous system abnormalities 
in early-onset type 1 diabetes mellitus. J Pediatr 153:385-390, 2008
3. DCCT: Hypoglycemia in the Diabetes Control and Complications Trial. The Diabetes Control 
and Complications Trial Research Group. Diabetes 46:271-286, 1997
4. Flykanaka-Gantenbein C: Hypoglycemia in childhood: long-term effects. Pediatr Endocrinol 
Rev 1 Suppl 3:530-536, 2004
5. Kodl CT, Seaquist ER: Cognitive dysfunction and diabetes mellitus. Endocr Rev 29:494-511, 
2008
6. DCCT: The effect of intensive treatment of diabetes on the development and progression of 
long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and 
Complications Trial Research Group. N Engl J Med 329:977-986, 1993
7. UKPDS: Intensive blood-glucose control with sulphonylureas or insulin compared with 
conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). 
UK Prospective Diabetes Study (UKPDS) Group. Lancet 352:837-853, 1998
8. Cryer PE: Banting Lecture. Hypoglycemia: the limiting factor in the management of IDDM.
Diabetes 43:1378-1389, 1994
9. Amiel SA, Tamborlane WV, Simonson DC, Sherwin RS: Defective glucose counterregulation 
after strict glycemic control of insulin-dependent diabetes mellitus. N Engl J Med 316:1376- 
1383, 1987
10. Amiel SA, Sherwin RS, Simonson DC, Tamborlane WV: Effect of intensive insulin therapy on 
glycemic thresholds for counterregulatory hormone release. Diabetes 37:901-907, 1988
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11. Mason GF, Rothman DL: Basic principles of metabolic modeling of NMR (13)C isotopic 
turnover to determine rates of brain metabolism in vivo. Metab Eng 6:75-84, 2004
12. Lin A, Ross BD, Harris K, Wong W: Efficacy of proton magnetic resonance spectroscopy in 
neurological diagnosis and neurotherapeutic decision making. NeuroRx 2:197-214, 2005
13. Cerdan S, Kunnecke B, Seelig J: Cerebral metabolism of [1,2-13C2]acetate as detected by in 
vivo and in vitro 13C NMR. J Biol Chem 265:12916-12926, 1990
14. Lebon V, Petersen KF, Cline GW, Shen J, Mason GF, Dufour S, Behar KL, Shulman GI, 
Rothman DL: Astroglial contribution to brain energy metabolism in humans revealed by 13C 
nuclear magnetic resonance spectroscopy: elucidation of the dominant pathway for 
neurotransmitter glutamate repletion and measurement of astrocytic oxidative metabolism. J 
Neurosci 22:1523-1531, 2002
15. Sibson NR, Mason GF, Shen J, Cline GW, Herskovits AZ, Wall JE, Behar KL, Rothman DL, 
Shulman RG: In vivo (13)C NMR measurement of neurotransmitter glutamate cycling, 
anaplerosis and TCA cycle flux in rat brain during. J Neurochem 76:975-989, 2001
16. Melo TM, Nehlig A, Sonnewald U: Metabolism is normal in astrocytes in chronically epileptic 
rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy. J 
Cereb Blood Flow Metab 25:1254-1264, 2005
17. Serres S, Bouyer JJ, Bezancon E, Canioni P, Merle M: Involvement of brain lactate in neuronal 
metabolism. NMR Biomed 16:430-439, 2003
18. Mason GF, Petersen KF, Lebon V, Rothman DL, Shulman GI: Increased brain monocarboxylic 
Acid transport and utilization in type 1 diabetes. Diabetes 55:929-934, 2006
19. Pan JW, Telang FW, Lee JH, de Graaf RA, Rothman DL, Stein DT, Hetherington HP: 
Measurement of beta-hydroxybutyrate in acute hyperketonemia in human brain. J Neurochem 
79:539-544, 2001
20. Herzog, R.I., et al., Lactate preserves neuronal metabolism and function following antecedent 
recurrent hypoglycemia. J Clin Invest, 2013. 123(5): p. 1988-98.
21. De Feyter, H.M., et al., Increased Brain Lactate Concentrations without Increased Lactate 
Oxidation during Hypoglycemia in Type 1 Diabetic Individuals. Diabetes, 2013.
4.
Research Plan: Summarize the study design and research procedures using non-technical language that can be 
readily understood by someone outside the discipline. Be sure to distinguish between standard of care vs. 
research procedures when applicable, and include any flowcharts of visits specifying their individual times and 
lengths.
Our goal is to measure the uptake kinetics and overall contribution of different neuron- 
specific energy substrates to brain neurotransmitter metabolism in type 1 diabetic and 
normal control subjects under hypoglycemia. For that purpose we will compare brain 
metabolite concentrations and metabolic fluxes by in vivo NMR spectroscopy in healthy 
controls to that of intensively treated type 1 diabetic patients with a history of severe 
hypoglycemia and hypoglycemia unawareness during a controlled hypoglycemic state, 
which we will induce by a hyperinsulinemic-hypoglycemic clamp.
To measure the effect of supplementation with different energy substrates and glucose 
itself on cognitive function subjects will undergo a 2-step hyperinsulinemic eu- and
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hypoglycemic clamp on two separate occasions, i.e. during a concomitant alternate fuel 
or labeled glucose or saline (control) infusion in random order. During each experimental 
condition the participants will complete a battery of validated cognitive tests that are 
sensitive to the cognitive impairment typically caused by hypoglycemia that we used 
previously in the context of hypoglycemia: Tests of non-memory function included digit 
symbol substitution, Tests of Everyday Attention, telephone book searching, and map 
searching in 1 and 2min. Working memory will be assessed by modified versions of the 
standard Wechsler Memory Scale Digit Span and Letter/Number Sequencing Tests, and 
map searching.
To define the effect of alternate fuels on brain neurotransmitter metabolism in 
hypoglycemia unawareness we will compare its ability to modulate the oxidation of 
infused stable isotope-labeled glucose in healthy adults with that in age- and weight 
matched subjects with c-peptide-negative T1DM with a history of hypoglycemia 
unawareness. During clamped hypoglycemia all subjects will undergo an NMR 
spectroscopy scan that permits detection of the stable isotope used, which will be either 
carbon13 (13C) or deuterium (2H). Using isotopically labeled-glucose as a tracer together 
with a co-infusion of 350mM unlabeled lactate, we will measure neuronal glucose 
metabolite enrichment, visualize them in three dimensions and determine the time 
courses of enrichment. These will be fitted with our metabolic model to determine whether 
increased lactate stimulates PDH flux and glucose oxidation under hypoglycemia and to 
what degree this varies across different regions of the brain.
Subject Selection and Preparation:
Two groups of subjects aged 18-65 years will be studied:
1) Group 1 will be T1DM subjects (C-peptide negative, HbA1c <7.5%) with a history of 
severe hypoglycemia and hypoglycemia unawareness as assessed by the Ryan 
HYPO Score (see below) and as evidenced by interview, questionnaire and glucose 
log and/or continuous glucose monitoring.
2) Group 2 will be non-diabetic healthy subjects (fasting plasma glucose < 100 mg/dL, 
normal HbA1c) who are matched for age and gender to the type 1 diabetic subjects, 
to serve as controls for the study.
The Ryan HYPO score:
During a 4-week period subjects record blood sugar levels 5 times per day. On the 
occasion that the glucose recorded is <54 mg/dl subjects are asked to describe the 
details of the event using a questionnaire recently developed at Edmonton by Ryan et 
al. to more objectively define hypoglycemia severity. Particular emphasis in the 
questionnaire is devoted to the type of symptoms and whether help was required to
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reverse the hypoglycemic event. From these data a composite HYPO score is 
generated based on the glucose readings and the patient’s reported hypoglycemic 
events. A composite score in the upper quartile of T1DM control patients will be 
required for enrollment in this study.
Subjects will be placed into one of following cohorts:
Cohort 1: Subjects from group 1 and group 2 will be scheduled for three study sessions 
as described below. A screening visit, an MR spectroscopy scan without 
hyperinsulinemic clamp (euglycemia) and an MR spectroscopy scan during a 
hyperinsulinemic clamp (hypoglycemia) with a tracer infusion.
Cohort 2: Subjects from group 1 and group 2 will be scheduled for two study sessions. A 
screening visit as described below. An MR spectroscopy scan that will be conducted 
during a hyperinsulinemic clamp (combined eu- and hypoglycemia) together with a 
tracer infusion.
Cohort 3: Subjects from group 1 and group 2 will be scheduled for two study sessions. A 
screening visit as described below. An MR spectroscopy scan conducted during a 
hyperinsulinemic clamp (combined eu- and hypoglycemia) without a tracer infusion.
Cohort 4: Healthy subjects from group 2 will be scheduled for one study session. They 
will undergo an MR spectroscopy scan without a hyperinsulinemic clamp nor any other
i.v. infusions (i.e. no i.v. infusions at all).
Cohort 5: Subjects from group 1 and group 2 will be scheduled for two study sessions. A 
screening visit as described below and a hyperinsulinemic clamp together with a non- 
labeled ketone or saline infusion without an MR spectroscopy scan.
Session A: Screening visit (YNHH Clinical Research Unit):
At the screening visit, study subjects in both the control and T1DM groups will complete 
the informed consent form for participation in voluntary clinical research. Demographic 
and clinical data collected will be reviewed by study personnel to confirm eligibility.
Once consented, all subjects will be further screened with a medical history & physical 
examination, blood work (electrolytes, hematocrit, creatinine, liver function tests and 
HbA1c) and a 12-lead electrocardiogram (EKG). Detailed subject inclusion and exclusion 
criteria are listed below (Human subjects – Subject recruitment). Subjects in the T1DM 
group only will be instructed to record their glucose readings to determine degree and 
frequency of hypoglycemia as described above. The subject will perform the preliminary 
cognitive tests at this point if they are selected for Cohort 5 at this point. The two 
subsequent MR sessions to complete the study will be scheduled at this point. The subject 
in the control group will have their two MR sessions scheduled without having to record 
any glucose readings.
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Session B: NMR spectroscopy without tracer infusion (Magnetic Resonance 
Research Center)
Subjects may be scheduled to arrive on the evening before the study if arriving early will 
be a difficulty. If such a case occurs, subjects will be admitted to the HRU for an overnight 
stay to be there for the morning without any delay.
On the morning of study, subjects will arrive directly at the MRRC. A member of the 
research team will verify that if the subject is from the T1DM group , the diabetic subject 
(as previously instructed) has taken his/her normal morning dose of insulin (or has 
continued to wear the insulin pump). This will not apply to subjects that are from the 
control group of non-diabetics participants. A urine pregnancy test will be completed on 
all female subjects of child bearing potential. Any female testing positive will be excluded 
from the study. All subjects will meet the staff, study procedures will be reviewed, and 
they will be exposed to the instrumentation and then placed in the scanner for NMR 
spectroscopy.
Imaging Procedures with the 4T/ 7T Bruker Advance NMR spectrometer (Bruker 
Instruments, Inc, Bellerica, MA) with a 94 cm bore: Proton MRS will be performed using 
the outer 1H decoupling coils of the 13C probe at 4T. Parameters and pulse sequences 
for GABA and glutamate acquisition will be the standard ones presently being established 
for this coil by Drs Mason and Rothman: Subjects will lie supine with the occipital cortex 
against a 7-cm surface coil tuned to 1H-MRS frequency of 170.4 MHz. T1-weighted 
images 30 seconds in duration will be acquired for subject positioning. 1st and 2nd order 
shimming will be performed automatically using the FASTERMAP procedure. We will then 
proceed to acquiring proton NMR spectra from a volume adjacent to the NMR coil for a 
period of 60 minutes. Total and regional power deposition will be below the FDA safety 
limits for RF power deposition. For 7T studies the pulse sequence will be performed using 
the multi Tx coil using pulse sequences and parameters established by Dr de Graaf, which 
will be verified regarding local power deposition using RF simulations. The 7T studies will 
be exploratory.
Session C: NMR spectroscopy with 13C/ 2H -tracer infusion (Magnetic Resonance 
Research Center)
Non-diabetic control subjects will be asked to remain fasting from midnight prior to the 
study. Upon arrival to the MRRC, their blood glucose will be checked and an IV catheter 
will be inserted for infusion of insulin and glucose to maintain normal glucose levels and 
the study will ensue as described below.
For type 1 diabetic subjects receiving insulin via an insulin pump , they will be asked to 
check their blood glucose at home before bed and on awakening to ensure that the 
glucose level is above 70mg/dl and below 200mg/dl. They will be instructed to call Dr. 
Herzog with any readings outside of this range. Upon arrival to the MRRC, their blood 
glucose will be checked as a safeguard, and insulin received via the insulin pump will be 
held. An IV catheter will be inserted for infusion of insulin and glucose to maintain normal 
glucose levels and the study will ensue as described below.
For type 1 diabetic subjects using multiple injections of insulin per day will be asked to 
ensure that the glucose level is above 70mg/dl and below 200mg/dl. As a safeguard, they
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will be asked to check their 
blood 
glucose 
at 
home 
before 
bed 
and 
on 
awakening. Upon arrival to 
the 
MRRC, 
their 
blood 
glucose will be checked; a 
nurse 
will 
place 
an 
intravenous (IV) line to start 
infusions of glucose and 
insulin to maintain normal 
glucose levels.
At 9:00 AM on the day of this 
NMR session, a standard IV 
catheter will be inserted and 
used to deliver a continuous
insulin infusion (2mU/kg/min) and a variable infusion of 20% dextrose. In addition a 
second IV line will be placed in the opposite arm to receive a normal saline solution and 
provide access for obtaining blood samples.
Once subjects arrive in the MR center they will be brought to a hypoglycemic state. The 
plasma glucose level will be gently lowered to 50  3 mg/dL (see blood draw schedule 
below).
Once hypoglycemia is achieved tracer will be administered (see Figure above). 
Depending on the cohort assignment this will involve one of the following infusions:
a) A 200mmol/l 13C /2H- alternate substrate infusion, which will be delivered through 
a port in the already-established IV site at a rate of 22µM/kg/min.
or
b) A 20% [1-13C]/ [2H]-glucose infusion at 
the same rate as the 20% unlabeled 
glucose infusion that is part of the 
ongoing    
hyperinsulinemic    
clamp. 
This ‘clamp glucose’ infusion will be held 
for the duration of the 13C/ 2H -glucose 
tracer infusion. In addition to the 13C/ 2H
-glucose infusion subjects will receive a 
simultaneous 
infusion 
of 
unlabeled 
350mM lactate (see adjacent Figure).
Subjects will be moved into the scanner for
magnetic resonance spectroscopy (MRS) studies. During the spectroscopy studies, 
subjects will lie supine in a 4.0 T 94 cm bore magnet. The back of each subject’s head
carbon-13 tracer infusion
30min
120min
Set up 1H
MRS 13C MRS
MRS study design. After a set up period of approximately 30 
minutes that includes a 1H MRS scan, subjects will be infused for 
120minutes with the isotopic labeled substrate.
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will be superjacent to an 8 cm diameter 13C/2H-surface coil receiver with a half quadrature 
coil 1H decoupling channel. Before the 13C/2H MRS measurements, an MRI will be 
obtained for localization purposes. From the image, a 48 cubic centimeter volume in the 
medical occipital-parietal lobe will be selected to obtain localized 13C/2H MRS spectra. 
The Bo homogeneity in the selected volume will be reduced to less than 0.08 ppm using 
automated shimming. Following shimming, localized MRS will be performed to the steady 
state 13C/2H-concentrations and fractional enrichments of 13C/2H-tracer, and the 2, 3, and 
4 positions of aspartate, glutamate, and glutamine. Throughout the study, samples of 
plasma will be obtained for measurement of glucose, insulin, ketones, lactate, 13C 
enrichment metabolites, and counterregulatory hormones (glucagon, epinephrine, and 
norepinephrine, cortisol). (See sample collection table Appendix A.) In addition, 
autonomic and neuroglycopenic symptoms will be assessed.
When the study is completed, subjects will receive a bolus and infusion of 10% dextrose 
to restore normoglycemia; they will then return to the HRU, where they will eat lunch. 
Once each subject appears clinically well with stable blood glucose levels above 70 
mg/dL for at least 60 minutes, the IV cannula can be removed and providing they are 
considered stable by the study physician, they can be discharged from the HRU.
Session D: Hyperinsulinemic Clamp with Cognitive Testing (Hospital Research 
Unit)
Non-diabetic control subjects will be asked to remain fasting from midnight prior to the 
study. Upon arrival to the HRU, their blood glucose will be checked and an IV catheter 
will be inserted for infusion of insulin and glucose to maintain normal glucose levels and 
the study will ensue as described below.
For type 1 diabetic subjects receiving insulin via an insulin pump will be asked to 
decrease their basal insulin rate by ~15% at 9 pm the night prior to the study. As a 
safeguard, they will be asked to check their blood glucose at home before bed and on 
awakening to ensure that the glucose level is above 70mg/dl and below 200mg/dl. They 
will be instructed to call Dr. Herzog with any readings outside of this range. Upon arrival 
to the HRU, their blood glucose will be checked as a safeguard, and insulin received via 
the insulin pump will be held. An IV catheter will be inserted for infusion of insulin and 
glucose to maintain normal glucose levels and the study will ensue as described below.
For type 1 diabetic subjects using multiple injections of insulin per day will be asked to 
ensure that the glucose level is above 70mg/dl and below 200mg/dl. They will be 
instructed to call Dr. Herzog with any readings outside of this range, halve their evening 
dose of long-acting insulin before going to bed and to skip their morning insulin injection. 
As a safeguard, they will be asked to check their blood glucose at home before bed and 
on awakening. Upon arrival to the HRU, their blood glucose will be checked; a nurse will 
place an intravenous (IV) line to start infusions of glucose and insulin to maintain normal 
glucose levels.
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Hyperinsulinemic clamp and cognitive testing. At 9:00 AM on the day of this 
hyperinsulinemic clamp session, a standard IV catheter will be inserted and used to 
deliver a continuous insulin infusion (2mU/kg/min) and a variable infusion of 20% 
dextrose. In addition a second IV line will be placed in the opposite arm to receive a 
normal saline solution and provide access for obtaining blood samples. During the clamp 
procedure the plasma glucose level will be gently lowered from the initial level of 95- 
100mg/dl to the target of 50  3 mg/dL.
Once a stable glycemic state is reached, subjects will receive a bolus-continuous infusion 
of 40µmol/min/kg unlabeled BHB for 5 min followed by a 12µmol/min/kg of BHB or 
infusion of a comparable volume of normal saline to control for possible volume effects 
for the remainder of the study.
Then a cognitive testing battery is administered via questionnaire as done by our group 
before (HIC#0505000079; PI: R. Sherwin). Tests of non-memory function included digit 
symbol substitution, Tests of Everyday Attention, telephone book searching, and map 
searching in 1 and 2min. Tests of immediate and delayed verbal memory and verbal 
memory recognition are adapted from the Wechsler Memory Scale logical memory tests. 
Working memory will be assessed by modified versions of the standard Wechsler Memory 
Scale Digit Span and Letter/Number Sequencing Tests, and map searching.
Throughout, blood is obtained for glucose, BHB, lactate, insulin, glucagon, cortisol, and 
catecholamines and will be assayed using established RIA, HPLC & ELISA methods in 
the YCCI Core Lab (see blood draw schedule below).
Blood Draw Schedule
start 
hypo 
clamp
glucose
~55mg/dl
for ~10-15min
glucose
~55mg/dl
for ~20-25min
Timeline
-60 min
-10 min
13C/2H-tracer
Baseline
start infusion*
+5
+10
+15
+30
+60
+90
+120
Insulin
X
X
X
X
X
X
Ketones
X
X
X
X
X
X
Lactate
X
X
X
X
X
X
X
C13 /2H
enrichment 
(during tracer 
infusion study)
X
X
X
X
X
X
X
X
Counterreg. 
hormone
X
X
X
X
X
X
glucose
--------------------------------glucose every 5 min during hypo clamp---------------------------------
* blood draws timed to alternate fuel infusion
Counter-regulatory hormones = E, NE, glucagon, cortisol, growth hormone
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In the event that scheduling issues preclude the sequencing of scanning sessions as 
described above, certain modifications can be made to accommodate a subject.
Blood Volumes and Schedule of Events Tables for each group are included at the end 
of this application.
5.
Genetic Testing
N/A 
A.
Describe
i.
the types of future research to be conducted using the materials, specifying if immortalization of 
cell lines, whole exome or genome sequencing, genome wide association studies, or animal 
studies are planned
ii.
the plan for the collection of material or the conditions under which material will be received
iii. the types of information about the donor/individual contributors that will be entered into a 
database
iv. the methods to uphold confidentiality
B.
What are the conditions or procedures for sharing of materials and/or distributing for future research 
projects?
C.
Is widespread sharing of materials planned?
D.
When and under what conditions will materials be stripped of all identifiers?
E.
Can donor-subjects withdraw their materials at any time, and/or withdraw the identifiers that connect them 
to their materials?
i.
How will requests to withdraw materials be handled (e.g., material no longer identified: that is, 
anonymized) or material destroyed)?
F.
Describe the provisions for protection of participant privacy
G. Describe the methods for the security of storage and sharing of materials
6.
Statistical Considerations: Describe the statistical analyses that support the study design.
Sample size calculations were performed using PASS 2002 (Hintze J. 2001. NCSS and 
PASS Number Cruncher Statistical Systems. Kaysville, Utah.). In our preliminary 
magnetic resonance spectroscopy experiments (see preliminary studies), under 
hypoglycemic conditions, we observed significantly greater fractions of glial oxidative 
metabolism supplied by acetate in T1DM subjects compared to non-diabetic controls 
(0.19  0.066 vs 0.10  0.020; a 90% increase in diabetics compared to controls). We will 
expect differences between severe hypoglycemia unaware and control subjects in this 
study to be on a similar scale. With a pooled standard deviation of 0.033, our proposed 
sample sizes (17 diabetic subjects and 17 non-diabetic controls) will provide 80% power 
at the two-sided 0.05 significance level to detect differences of 0.033 between diabetic 
subjects and non-diabetic controls representing a 33% increase from non-diabetic 
subjects. In short, we should easily have sufficient numbers of to address our primary 
endpoint of enhanced ketone body uptake and metabolism following recurrent 
hypoglycemia. Using this data, our proposed sample sizes of 17 subjects per group will 
provide >80% power to detect a 20% difference in the severe hypoglycemia unaware 
compared to each control group. We anticipate the changes seen will be much greater 
than that. Because small effects cannot be detected with sufficient power, conclusions 
based on failure to reject the null hypothesis will be avoided.
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The same group size considerations apply to the cohorts studied with glucose/lactate co- 
infusions and the cognitive performance analysis under ketone infusion outside of the 
MRI scanners.
Data analysis will be performed using SAS statistical software version 8.2. Prior to the 
fundamental analysis, all data will be appropriately screened for accuracy, missing data, 
outliers and agreement with assumptions for the analyses. Univariate descriptive 
statistics and plots will first be employed to determine out-of-range values as well as 
plausible means and standard deviations. Non-normal variables will be identified through 
the inspection of normal probability plots and with the Kolmogorov-Smirnov test. If 
required, appropriate transformations or non-parametric tests will be applied to variables 
in violation of the model assumptions. James Dziura, Ph.D., the GCRC biostatistician, is 
available for advice in data analysis.
To test the hypothesis that patients with hypoglycemia unawareness exhibit enhanced 
CNS ketone body metabolism we will assess brain beta-hydroxybutyrate transport and 
its absolute levels and the ratio of brain beta-hydroxybutyrate contribution to the neuronal 
and astroglial TCA cycle. In addition, we will determine the absolute concentration of brain 
neurotransmitter levels using proton NMR. Group comparisons will be assessed by 
analysis of variance (ANOVA) for repeated measures. Data will be tested for multisample 
sphericity using Mauchly’s and Box tests. If the assumption of homogenous covariance 
matrices is violated a covariance pattern model will be employed to allow for separate 
sets of covariance parameters for type 1 diabetic vs. nondiabetic controls. Evaluation of 
a significant diagnosis by clamp condition interaction will be determined by paired or 
unpaired t-tests (for within and between subjects factors respectively), using the 
interaction variance term from the ANOVA with a Holm correction for multiple testing.
7. Subject classification: Check off all classifications of subjects that will be targeted for enrollment in the 
research project. Will subjects who may require additional safeguards or other considerations be enrolled in the 
study? If so, identify the population of subjects requiring special safeguards and provide a justification for their 
involvement.
Children
Non-English Speaking 
Decisionally Impaired
Healthy 
Prisoners 
Employees
Fetal material, placenta, or dead fetus 
Economically disadvantaged persons 
Pregnant women and/or fetuses
Yale Students
Females of childbearing potential
NOTE: Is this research proposal designed to enroll children who are wards of the state as potential subjects? 
 No (If yes, see Instructions section VII #4 for further requirements)
8. Inclusion/Exclusion Criteria: What are the criteria used to determine subject inclusion or exclusion?
Inclusion criteria for subjects:
1.Type 1 diabetes mellitus of > 5 years (C-peptide negative age 18-60 years, on 
intensive insulin therapy (HbA1c <7.5%)
2. Non-diabetic volunteers, age 18-65 years that do not meet any of the exclusion 
criteria.
Yes
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The following criteria will exclude subjects from study:
1. Pregnancy
2. Significant baseline anemia (hemoglobin <11.0 or hematocrit < 33%)
3. A history of liver cirrhosis or porto-caval shunt surgery.
4. Any contraindications for MRI scanning.
5. Subjects that follow a vegetarian diet
6. Subjects that exercise heavily on a regular basis (i.e. marathon runners)
7. Subjects with a history of anxiety/ panic attacks
9. How will eligibility be determined, and by whom?
Eligibility to participate in the study will be determined by Raimund Herzog, MD. 
Eligibility will be determined based on information collected at the screening visit after 
consent is obtained. This will include findings from physical examination, medical 
history, EKG, and laboratory testing in accordance with the inclusion and exclusion 
criteria listed above.
10.Risks: Describe the reasonably foreseeable risks, including risks to subject privacy, discomforts, or 
inconveniences associated with subjects participating in the research.
10.1. Intravenous catheters. The placement of intravenous catheters and/or the use of 
intravenous infusions can result in local hematoma formation or thrombophlebitis. 
Transient vasovagal symptoms including nausea, sweating, and lightheadedness 
may also occur during intravenous catheter placement.
10.2. Blood sampling. Same as above and frequent blood sampling may result in a drop 
in hematocrit.
10.3. Hyperinsulinemic 
hypoglycemic 
clamp 
technique. 
The 
hyperinsulinemic 
hypoglycemic clamp technique can result in hypoglycemia symptoms of varying 
severity. Subjects may experience autonomic symptoms including hunger, anxiety, 
tremor, palpitations, and/or diaphoresis. Others may experience fatigue, 
concentration difficulties, and/or mild confusion. The risk of plasma glucose levels 
falling below the target value of 50 ± 3 mg/dL is small but present; affected patients 
may experience exaggerated symptoms, as well as the development of dizziness, 
blurred vision, and/or confusion. All such symptoms would be rapidly reversible with 
an intravenous dextrose infusion.
10.4. Infusion of lactate. In some subjects with severe panic disorder lactate infusions at 
a lever higher than proposed here for MR purposes can precipitate a panic attack. 
In healthy subjects without panic attacks lactate infusion is not known to induce such 
attacks. For this reason we are screening potential study subjects for a past medical 
history of panic attacks and they will be excluded from participation in our study.
10.5. Magnetic resonance imaging/spectroscopy. Magnetic resonance (MR) is a 
technique that uses magnetism and radio waves, not x-rays, to take pictures and 
measure chemicals of various parts of the body. The United States Food and Drug
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Administration (FDA) has set guidelines for magnet strength and exposure to radio 
waves, and we carefully observe those guidelines.
The Subject will be watched closely throughout the MR study. Physiological 
monitoring will be done, including O2 saturation, pulse rate and blood pressure. 
Some subjects may feel uncomfortable or anxious. If this happens to the subject, 
the subject may ask the study nurse that is present in the scanner room to stop the 
study at any time and the MR staff will take the subject out of the MR scanner. On 
rare occasions, some subjects might feel dizzy, get an upset stomach, have a 
metallic taste or feel tingling sensations or muscle twitches. These sensations 
usually go away quickly however the subject will be instructed to please tell the 
research staff if they experience them. Dr. Herzog, who has admitting privileges at 
YNHH will be available by cell phone at all times surrounding a study.
There are some risks with an MR study for certain people. If the subject has a 
pacemaker or some metal objects inside their body, the subject may not be in this 
study because the strong magnets in the MR scanner might harm them. Another risk 
is a metallic object flying through the air toward the magnet and hitting the subject. 
To reduce this risk the MR staff requires that all people involved with the study 
remove all metal from their clothing and all metal objects from their pockets. Nothing 
metal can be brought into the magnet room at any time. Subjects will walk through 
a ferromagnetic detector. Also, once the subject is in the magnet, the door to the 
room will be closed so that no one from outside accidentally goes near the magnet.
The MR staff want the subject to read and answer very carefully the questions on 
the MR Safety Questionnaire related to the subject’s personal safety. The subject 
will be asked to take a moment, to be sure that they have read the MR Safety 
Questionnaire and be sure to tell the MR staff any information they think might be 
important.
This MR study is for research purposes only and is not in any way a clinical 
examination. The scans performed in this study are not designed to find 
abnormalities. The primary investigator, the lab, the MR technologist, and the 
Magnetic Resonance Research Center are not qualified to interpret the MR scans 
and are not responsible for providing a diagnostic evaluation of the images. If a 
worrisome finding is seen on the subject’s scan, a radiologist or another physician 
will be asked to review the relevant images. Based on his or her recommendation (if 
any), the primary investigator or consulting physician will contact the subject, inform 
the subject of the finding, and recommend that the subject seek medical advice as 
a precautionary measure. The decision for additional examination or treatment 
would lie solely with the subject and the subject’s physician. The investigators, the 
consulting physician, the Magnetic Resonance Research Center, and Yale 
University are not responsible for any examination or treatment that the subject 
receive based on these findings. The images collected in this study are not a clinical 
MR exam and for that reason, they will not be made available for diagnostic 
purposes.
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10.6 Stable isotope infusions: 13C/2H –labeled alternate energy substrates or 13C/2H-
glucose. Carbon-13 and 2H (deuterium) are stable isotopes of naturally occurring 
substances (carbon12 and hydrogen, respectively). 2H has been used as 
metabolic tracer in the early days of in vivo MRS, but was never as popular as 13C 
approaches. Recent technical developments have motivated us to explore the 
usefulness of 2H in metabolic imaging. There are no known side effects are 
associated with the administration of the labeled substrates acetate, BHB and 
lactate or glucose, all of which have been used by our group and colleagues at 
MRRC for many years. The MRRC has extensive experience with stable isotope 
infusions including acetate, BHB, glucose and lactate (HIC # 10605, Rothman PI)) 
and to date there have been no adverse events due to the infusion.
11. 11. Minimizing Risks: Describe the manner in which the above-mentioned risks will be minimized.
The primary risks to the subject are impact by magnetic objects accidentally brought into 
the magnet room, anxiousness, irritation due to the IV blood sampling, and hypoglycemia 
after the glucose infusion. The investigators in this protocol pioneered human infusion 
studies of stable isotope labeled metabolic substrates in MR systems and have been 
routinely performing these studies since 1988. Studies of brain metabolism using stable 
isotope infusion in the MRC have been performed since 1990. To date there have been 
no patient injuries during these studies and only minor adverse incidences (anxiety, IV 
irritation).
To confront the specific risks listed above:
11.1 Intravenous catheters will be placed under sterile conditions by experienced HRU 
staff members. All infused solutions will be prepared in sterile form by the YNHH 
pharmacy and will be tested for pyrogenicity and sterility prior to use. During 
dextrose infusions, 20% dextrose will be used to reduce the risk of thrombophlebitis 
normally associated with the use of excessively hypertonic glucose solutions.
11.2 No subject with a hematocrit less than 37% for males and less than 33% for females 
will be studied. All subjects having donated blood within 30 days of the study will be 
asked to postpone study participation (with a repeat blood count prior to future 
enrollment), and patients will be advised to refrain from blood donation for 30 days 
after each study completion. Total blood loss will not exceed half the amount of a 
regular blood donation for each study group. All blood losses will be replaced 
quantitatively with normal saline.
11.3 All study procedures, including the hyperinsulinemic clamp, will be performed under 
the direct supervision of a qualified, licensed practitioner.
11.4 No subject with a history of panic attacks will be enrolled in our study. To identify 
these subjects specific screening questions are administered during the screening 
visit. Licensed physicians and study nurses are present throughout the study to 
immediately recognize any distress subjects may experience. Studies would be 
immediately terminated should despite careful screening such an event still occur.
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11.5 To avoid excess hypoglycemia during the hyperinsulinemic clamp, plasma glucose 
will be checked every five minutes using a bedside glucose monitor. Based on these 
measurements, the plasma glucose will be adjusted via the D20 infusion.
11.6 All potential study subjects will also be screened for MR safety using the standard 
MR safety protocol developed by Yale New Haven Hospital’s Diagnostic Imaging 
department. Subjects with claustrophobia or with contraindications to MR scanning 
will be excluded from the study. The MR safety screening process will be performed 
both during the screening interview and on the day of an MR study.
11.7 To reduce MRI risks we require that all people involved with the study remove all 
metal from their clothing and all metal objects from their pockets. Nothing metal can 
be brought into the magnet room at any time. Subjects will also walk through a 
ferromagnetic detector. Also, once subjects are in the magnet, the door to the room 
will be closed so that no one from outside accidentally goes near the magnet. In 
addition, patients can stop study at any time if anxiety is a significant issue.
11.8 To reduce the risk of injury in the MRI scanner, subjects will go through a 
ferromagnetic metal detector, to prevent any metal objects being taken into the 
scanner.
11.9 Stable isotope infusions will be prepared in sterile form by the YNHH pharmacy and 
will be tested for pyrogenicity and sterility prior to use.
11.10 Study subjects will be closely followed by an experienced clinical research team, 
including an experienced nurse with ACLS training who is with a patient at all times, 
a licensed MD or practitioner, and an experienced MRS technologist. All personnel 
involved in the study will be trained in MR safety procedures. The MD or licensed 
practitioner and technologist who are outside of the room have direct visual contact 
with the subject and nurse through a window, a TV monitor, and audio contact 
through a speaker system. Subjects who become claustrophobic in the magnet will 
be removed immediately from the magnet room. Patients who experience distress 
in the magnet room for any reason including seizures, loss of consciousness, or pain 
will be removed from the magnet room in accordance with well-established 
procedures developed for the Magnetic Resonance Research Center. Should an 
emergency arise, the MR technologist will call 911 and have an ambulance with 
personnel trained in resuscitation sent to the MR Center. The other members will 
remove the patient via the detachable patient bed from the magnet room If 
immediate resuscitation is needed the physician present during the study will 
perform this procedure, using a fully equipped crash cart which will be in the console 
room during the study.
All subjects will be given a telephone number to enable immediate communication with 
the P.I. or one of the co-investigators, to report any delayed adverse effects and to 
receive recommendations for treatment of any adverse effects.
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12. Data and Safety Monitoring Plan: Include an appropriate Data and Safety Monitoring Plan (DSMP) based on 
the investigator’s risk assessment stated below. (Note: the HIC will make the final determination of the risk to 
subjects.) For more information, see the Instructions, page 24.
a.
What is the investigator’s assessment of the overall risk level for subjects participating in this 
study?
Moderate
b.
If children are involved, what is the investigator’s assessment of the overall risk level for the 
children participating in this study?
N/A
c.
Copy, paste, and then tailor an appropriate Data and Safety Monitoring Plan 
from http://www.yale.edu/hrpp/forms-templates/biomedical.html for
i.
Minimal risk
ii.
Greater than minimal/moderate risk
iii.
High risk
The risks associated with the current study are deemed moderate for the following 
reasons:
We do not view the risks associated with the combined use of insulin and dextrose as 
minimal. Given our experience with the combined co-administration of insulin and 
dextrose, we do not view the proposed studies as high risk.
Although we have assessed the proposed study as one of moderate risk, the potential 
exists for anticipated and/or unanticipated adverse events, serious or otherwise, to occur 
since it is not possible to predict with certainty the absolute risk in any given individual or 
in advance of first-hand experience with the proposed study methods.
Therefore, we provide a plan for monitoring the data and safety of the proposed study as 
follows:
Attribution of Adverse Events:
Adverse events will be monitored for each subject participating in the study and 
attributed to the study procedures / design by the principal investigator Dr Herzog 
according to the following categories:
a.) Definite: Adverse event is clearly related to investigational procedures. 
b.) Probable: Adverse event is likely related to investigational procedures. 
c.) Possible: Adverse event may be related to investigational procedures.
d.) Unlikely: Adverse event is likely not to be related to the investigational 
procedures.
e.) Unrelated: Adverse event is clearly not related to investigational procedures.
Plan for Grading Adverse Events:
The following scale will be used in grading the severity of adverse events noted during 
the study:
1. Mild adverse event
2. Moderate adverse event
3. Severe
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Plan for Determining Seriousness of Adverse Events:
Serious Adverse Events:
In addition to grading the adverse event, the PI will determine whether the adverse event 
meets the criteria for a Serious Adverse Event (SAE). An adverse event is considered 
serious if it:
1. is life-threatening
2. results in in-patient hospitalization or prolongation of existing hospitalization
3. results in persistent or significant disability or incapacity
4. results in a congenital anomaly or birth defect OR
5. results in death
6. based upon appropriate medical judgment, may jeopardize the subject’s health 
and may require medical or surgical intervention to prevent one of the other 
outcomes listed in this definition, or
7. adversely affects the risk/benefit ratio of the study
An adverse event may be graded as severe but still not meet the criteria for a Serious 
Adverse Event. Similarly, an adverse event may be graded as moderate but still meet the 
criteria for an SAE. It is important for the PI to consider the grade of the event as well as 
its “seriousness” when determining whether reporting to the HIC or HSC is necessary.
Plan for reporting serious AND unanticipated AND related adverse events, 
anticipated adverse events occurring at a greater frequency than expected, and 
other unanticipated problems involving risks to subjects or others to the HIC or 
HSC.
13.The investigator will report the following types of adverse events to the HIC or  HSC:
a) serious AND unanticipated AND possibly, probably or definitely related events;
b) anticipated adverse events occurring with a greater frequency than expected; and
c)
other unanticipated problems involving risks to subjects or others.
These adverse events or unanticipated problems involving risks to subjects or others will 
be reported to the HIC or HSC within 48 hours of it becoming known to the investigator, 
using the appropriate forms found on the website.
Plan for reporting adverse events to co-investigators on the study, as appropriate 
the protocol’s research monitor(s), e.g., industrial sponsor, Yale Center for Clinical 
Investigation Research Subject Advocates (RSAs), Cancer Center's Quality 
Assurance, Compliance and Safety Committee (QUACS) Protocol Review 
Committee (PRC), DSMBs, study sponsors, funding and regulatory agencies, and 
regulatory and decision-making bodies.
For the current study, the following individuals, funding, and/or regulatory 
agencies will be notified (choose those that apply):
-
All Co-Investigators listed on the protocol.
-
Yale Center for Clinical Investigation Research Subject Advocates (RSAs)
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The principal investigator Dr Herzog will conduct a review of all adverse events upon 
completion of every study subject. The principal investigator will evaluate the frequency 
and severity of the adverse events and determine if modifications to the protocol or 
consent form are required.
d. For multi-site studies for which the Yale PI serves as the lead investigator:
i.
How will adverse events and unanticipated problems involving risks to subjects or others be 
reported, reviewed and managed?
ii.
What provisions are in place for management of interim results?
iii.
What will the multi-site process be for protocol modifications?
N/A
12. Statistical Considerations: Describe the statistical analyses that support the study design.
Sample size calculations were performed using PASS 2002 (Hintze J. 2001. NCSS and 
PASS Number Cruncher Statistical Systems. Kaysville, Utah.). In our preliminary 
magnetic resonance spectroscopy experiments (see preliminary studies), under 
hypoglycemic conditions, we observed significantly greater fractions of glial oxidative 
metabolism supplied by acetate in T1DM subjects compared to nondiabetic controls (0.19
 0.066 vs 0.10  0.020; a 90% increase in diabetics compared to controls). We will expect 
differences between severe hypoglycemia unaware and control subjects in this study to 
be on a similar scale. With a pooled standard deviation of 0.033, our proposed sample 
sizes (17 diabetic subjects and 17 nondiabetic controls) will provide 80% power at the 
two-sided 0.05 significance level to detect differences of 0.033 between diabetic subjects 
and nondiabetic controls representing a 33% increase from nondiabetic subjects. In short, 
we should easily have sufficient numbers of to address our primary endpoint of enhanced 
ketone body uptake and metabolism following recurrent hypoglycemia. Using this data, 
our proposed sample sizes of 17 subjects per group will provide >80% power to detect a 
20% difference in the severe hypoglycemia unaware compared to each control group. 
We anticipate the changes seen will be much greater than that. Because small effects 
cannot be detected with sufficient power, conclusions based on failure to reject the null 
hypothesis will be avoided.
Data analysis will be performed using SAS statistical software version 8.2. Prior to the 
fundamental analysis, all data will be appropriately screened for accuracy, missing data, 
outliers and agreement with assumptions for the analyses. Univariate descriptive 
statistics and plots will first be employed to determine out-of-range values as well as 
plausible means and standard deviations. Non-normal variables will be identified through 
the inspection of normal probability plots and with the Kolmogorov-Smirnov test. If 
required, appropriate transformations or non-parametric tests will be applied to variables 
in violation of the model assumptions. James Dziura, Ph.D., the GCRC biostatistician, is 
available for advice in data analysis.
To test the hypothesis that patients with hypoglycemia unawareness exhibit enhanced 
CNS alternate fuel metabolism we will assess brain beta-hydroxybutyrate transport and 
its absolute levels and the ratio of brain alternate fuel contribution to the neuronal and 
astroglial TCA cycle. In addition, we will determine the absolute concentration of brain
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SECTION VI: RESEARCH INVOLVING DRUGS, BIOLOGICS, RADIOTRACERS, PLACEBOS AND
DEVICES
neurotransmitter lebels using proton NMR. Group comparisons will be assessed by 
analysis of variance (ANOVA) for repeated measures. Data will be tested for multisample 
sphericity using Mauchly’s and Box tests. If the assumption of homogenous covariance 
matrices is violated a covariance pattern model will be employed to allow for separate 
sets of covariance parameters for type 1 diabetic vs. nondiabetic controls. Evaluation of 
a significant diagnosis by clamp condition interaction will be determined by paired or 
unpaired t-tests (for within and between subjects factors respectively), using the 
interaction variance term from the ANOVA with a Holm correction for multiple testing.
If this section (or one of its parts, A or B) is not applicable, state N/A and delete the rest of the 
section.
A. DRUGS and BIOLOGICS
1.
Identification of Drug or Biologic: What is (are) the name(s) of the drug(s) or
biologic(s) being used? Identify whether FDA approval has been granted and for what indication(s).
[13C] / 2H -labeled BHB 
[13C] / 2H -labeled acetate 
[13C] / 2H -labeled lactate 
[13C] / 2H -labeled glucose 
Unlabeled BHB 
Unlabeled acetate 
Unlabeled lactate 
Unlabeled glucose
All protocols which utilize a drug or biologic not approved by, but regulated by, the FDA must provide the 
following information:
a. What is the Investigational New Drug (IND) number assigned by the FDA?
b. Who holds the IND?
Alternatively, an exemption from IND filing requirements may be sought for a clinical 
investigation of a drug product that is lawfully marketed in the United States. If there is no IND and 
an exemption is being sought, review the following categories and complete the category that applies 
(and delete the inapplicable categories):
Exempt Category 1
The clinical investigation of a drug product that is lawfully marketed in the United States can be 
exempt from IND regulations if all of the following are yes:
i. The intention of the investigation is NOT to report to the FDA as a well-controlled study in support 
of a new indication for use or to be used to support any other significant change in the labeling for 
the drug.
Yes
No
ii.
The drug that is undergoing investigation is lawfully marketed as a prescription drug product, and
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the intention of the investigation is NOT to support a significant change in the advertising for the 
product.
Yes
No
iii. The investigation does NOT involve a route of administration or dosage level or use in populations 
or other factor that significantly increases the risks (or decreases the acceptability of the risks) 
associated with the use of the drug product.
Yes
No
iv. The investigation will be conducted in compliance with the requirements for institutional (HIC)
review and with the requirements for informed consent of the FDA regulations (21 CFR Part 50 and 21 CFR 
Part 56).
Yes
No
v. The investigation will be conducted in compliance with the requirements regarding promotion and charging 
for investigational drugs.
Yes
No
Exempt Category 2 (all items i, ii, and iii must be checked to grant a category 2 exemption)
i. The clinical investigation is for an in vitro diagnostic biological product that involves one or 
more of the following (check all that apply):
Blood grouping serum 
Reagent red blood cells 
Anti-human globulin
ii. The diagnostic test is intended to be used in a diagnostic procedure that confirms the 
diagnosis made by another, medically established, diagnostic product or procedure; and
iii. The diagnostic test is shipped in compliance with 21 CFR §312.160.
Exempt Category 3
The drug is intended solely for tests in vitro or in laboratory research animals if shipped in accordance with 
21 CFR 312.60
Exempt Category 4
A clinical investigation involving use of a placebo if the investigation does not otherwise require submission 
of an IND.
2.
Background Information: Provide a description of previous human use, known risks, and data addressing 
dosage(s), interval(s), route(s) of administration, and any other factors that might influence risks. If this is the 
first time this drug is being administered to humans, include relevant data on animal models.
13C-labeled acetate, 13C-labeled BHB, 13C-labeled lactate and 13C-glucose have been 
administered by the PI and his mentors Drs Sherwin and Rothman in previous studies 
(HIC # 10605) without any side effects:
Alternate Substrate infusion rates consist of a bolus of 16.7ml/min for 20 minutes followed 
by   a   continuous   rate   of   0.77ml/min   for   an   additional   100   minutes.    
Glucose will be infused at the same rate as required to maintain a subject’s plasma 
glucose level at the target hypoglycemic level of 55mg/dl. Infusion of unlabeled lactate 
(350 mmol/L) will occur at a priming dose of 300mol/kg given over 5 min followed by a 
continuous infusion of 20mol/kg/min for 120min.
Of note, carbon-13 and deuterium are natural isotopes of carbon. They are non- 
radioactive and have no side effects.
3.
Source:
a) Identify the source of the drug or biologic to be used.
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The tracer-labeled energy substrates are purchased in powder form (from Sigma- 
Aldrich or Cambridge Isotopes, Inc.) and have been certified to be sterile and free 
of pyrogens. The glucose is made into a solution of 20gram/100 ml (D-20) by the 
Yale New Haven Hospital Investigational Drug Service (IDS). The solutions are 
tested to be free of pyrogens and to be sterile before being administered. All 
certifications are kept with the IDS.
Clinical grade labeled acetate, BHB and lactate are purchased in powder form 
(also from Sigma-Aldrich or Cambridge Isotopes, Inc.) which has been certified to 
be sterile and free of pyrogens. The acetate is made into a solution of 350mmol/liter 
by the Yale New Haven Hospital Investigational Drug Service (IDS). The solutions 
are tested to be free of pyrogens and to be sterile, and they have a neutral pH 
balance before being administered. All certifications are kept with the IDS.
b) Is the drug provided free of charge to subjects?
Yes
No
If yes, by whom?
N/A
4.
Preparation and Use: Describe the method of preparation, storage, stability information, and for parenteral 
products, method of sterilization and method of testing sterility and pyrogenicity.
Check applicable Investigational Drug Service utilized:
YNHH IDS
Yale Cancer Center
CMHC Pharmacy
West Haven VA
Other:
None
Note: If the YNHH IDS (or comparable service at CMHC or WHVA) will not be utilized, explain in detail 
how the PI will oversee these aspects of drug accountability, storage, and preparation.
The compounds will be sterility tested and certified to be free of pyrogens by the 
company. All certifications are kept in YNHH-IDS. YNHH-IDS prepares the study 
infusion the day before the study and the study infusate is kept at 4C.
5.
Use of Placebo: 
 Not applicable to this research project
If use of a placebo is planned, provide a justification which addresses the following:
a.
Describe the safety and efficacy of other available therapies. If there are no other 
available therapies, state this.
b. State the maximum total length of time a participant may receive placebo while on the study.
c.
Address the greatest potential harm that may come to a participant as a result of receiving 
placebo.
d. Describe the procedures that are in place to safeguard participants receiving placebo.
6.
Use of Controlled Substances:
Will this research project involve the use of controlled substances in human subjects?
Yes  
 No
See HIC Application Instructions to view controlled substance listings.
If yes, is the use of the controlled substance considered:
Therapeutic: The use of the controlled substance, within the context of the research, has the potential to 
benefit the research participant.
Non-Therapeutic: Note, the use of a controlled substance in a non-therapeutic research study involving 
human subjects may require that the investigator obtain a Laboratory Research License. Examples include
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SECTION VII: RECRUITMENT/CONSENT AND ASSENT PROCEDURES
controlled substances used for basic imaging, observation or biochemical studies or other non-therapeutic 
purposes. See Instructions for further information.
7.
Continuation of Drug Therapy After Study Closure   
 Not applicable to this project
Are subjects provided the opportunity to continue to receive the study drug(s) after the study has ended?
Yes If yes, describe the conditions under which continued access to study drug(s) may apply as well as 
conditions for termination of such access.
No If no, explain why this is acceptable.
B. DEVICES
1.
Are there any investigational devices used or investigational procedures performed at YNHH, e.g., 
YNHH Operating Room or YNHH Heart and Vascular Center? Yes
No 
1. Targeted Enrollment: Give the number of subjects:
1.1.
targeted for enrollment at Yale for this protocol 100_
1.2.
if this is a multi-site study, give the total number of subjects targeted across all sites n/a_
2. Indicate recruitment methods below. Attach copies of any recruitment materials that will be used.
Flyers
Internet/Web Postings
Posters
Mass E-mail Solicitation
Letter
Departmental/Center Website
Medical Record Review
Departmental/Center Research Boards
Departmental/Center Newsletters
Web-Based Clinical Trial Registries 
Other (describe): Yalestudies.org and yalestudies social media Facebook and Twitter 
Clinicaltrials.gov Registry (do not send materials to HIC)
Radio 
Telephone 
Television 
Newspaper
3.
Recruitment Procedures:
3.1.
Describe how potential subjects will be identified.
Some subjects will be identified through their previous participation HIC #1009007394: “Effect of a 
long-term Medium Chain Triglyceride diet on cognitive function and brain activation.” and HIC 
#0505000079: “Effect of Medium Chain Fatty Acids in cognitive function during acute hypoglycemia in 
patients with type 1 diabetes.”.
Possible study participants will be approached by their caregivers or study staff at the different locations 
where Type 1 Diabetic patients are seen at Yale SOM, YNHH and YMG. These sites include the Adult 
Endocrine clinic (Yale Diabetes Center, YNHH), the Yale Pediatric Endocrine Clinic, Veterans Affairs 
Hospital, West Haven, community clinics and Yale University Health. We will also accept referrals from 
outpatient clinics in the community. In addition, the diabetes registry will be used to identify eligible 
patients and contact them by phone or letter, since this category of patients have already given their 
consent to be contacted for potential trials research. Flyers will be put up on notice boards at and around 
Yale University and Yale New Haven Hospital. Study volunteer listings will be requested from the YCCI 
volunteer database (HIC#0805003779) to assist with recruitment of subjects. Also, the Diabetes Registry 
(HIC#0911005973) will be used to contact Type 1 Diabetic subjects to discuss enrollment into the study. 
Subjects will be contacted through email and telephone. Brief screenings will take place either over the 
telephone or email and subjects will then be invited to a full screening at the HRU.
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3.2.
Describe how potential subjects are contacted.
3.3.
Who is recruiting potential subjects?
Flyers will be put up on notice boards at and around Yale University and Yale New 
Haven Hospital. Study volunteer listings will be requested from the YCCI volunteer 
database (0805003779) to assist with recruitment of subjects. Also, the Diabetes 
Registry (HIC# 0911005973) will be used to contact Type 1 Diabetic subjects to 
discuss enrollment into the study. Subjects will be contacted through email and 
telephone. Brief screenings will take place either over the telephone or email and 
subjects will then be invited to a full screening at the HRU.
The text to appear on yalestudies.org is as follows:
Purpose (healthcare version): The goal of this study is to characterize the adaptations of brain 
metabolism in T1DM subjects exposed to frequent hypoglycemic episodes.
We will determine the contribution of alternate energy substrates to brain neurotransmitter 
metabolism under experimentally controlled low blood sugar levels. For purposes of this study 
we are looking to enroll healthy controls and T1DM subjects.
Purpose (subject friendly version): We are trying to understand how brain metabolism in 
T1DM subjects changes in order to develop therapies that can protect the brain from injury 
during events of extreme low blood sugar.
Inclusion Criteria: Type 1 Diabetes Mellitus >5 years duration 
HbA1c <7.5%
A person in good health
Exclusion Criteria:
1. Significant Systemic Disease
2. Pregnancy
3. Significant Anemia
4. Excessive Exercise
5. Vegetarian Diet
6. Excessive Alcohol Use 
Substance Abuse
Compensation offered:
Up to $350
4.
Screening Procedures
4.1.
Will email or telephone correspondence be used to screen potential subjects for eligibility prior to the 
potential subject coming to the research office? 
 Yes
No
Through telephone correspondence, potential subjects will be asked the following information: Name, age, 
gender, date of birth, place of birth, race/ethnicity, address, phone number, height, weight, history of 
diabetes including treatment and complications, most recent HbA1c level, medications, history of other 
medical problems, history of claustrophobia, vegetarian, right/left handed, and previous studies at Yale. 
This information will be protected according to HIPPA policies and will be destroyed if the individual is
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determined to not be eligible for the study during this telephone correspondence. The purpose and potential 
complications of the study will be explained to each subject in detail.
The actual screening visit cannot occur until the participant has signed the informed consent. During the 
screening visit, the informed consent form and study details are reviewed in detail by research staff and the 
subject will be asked to read the informed consent form (approved by the Yale Human Investigations 
committee). The subject will be given time to ask questions and only after that will the subject be asked to 
give written informed consent to participate.
At the screening visit, the participant will undergo the following assessments: physical examination with 
height and weight, medical history, blood draw for hematocrit, HbA1c levels, potassium, bicarbonate, 
creatinine, ALT/AST, lipid profile, ketones, and c-peptide. If female and of childbearing potential, a 
pregnancy test will be performed. Inclusion will be based on the above information and determined by the 
PI.
4.2.
If yes, identify below all health information to be collected as part of screening and check off any of the 
following HIPAA identifiers to be collected and retained by the research team during this screening 
process.
Prior to the potential subject traveling to Yale for the screening visits, a collection of information concerning 
their eligibility will be done to help determine if they are eligible to be screened for inclusion. This will be 
done only to prevent unnecessary screening and travel for those who can be determined to be ineligible 
based on recognizable exclusion criteria. Through telephone correspondence, potential subjects will be 
asked the following information: Name, age, gender, date of birth, place of birth, race/ethnicity, address, 
phone number, height, weight, history of diabetes including treatment and complications, most recent 
HbA1c level, medications, history of other medical problems, history of claustrophobia, vegetarian, 
right/left handed, and previous studies at Yale. This information will be protected according to HIPPA 
policies and will be destroyed if the individual is determined to be not be eligible for the study during this 
telephone correspondence.
HEALTH INFORMATION TO BE COLLECTED:
HIPAA identifiers:
Names
All geographic subdivisions smaller than a State, including: street address, city, county, precinct, zip codes and 
their equivalent geocodes, except for the initial three digits of a zip code if, according to the current publicly-available 
data from the Bureau of the Census: (1) the geographic unit formed by combining all zip codes with the same three 
initial digits contains more than 20,000 people, and (2) the initial three digits of a zip code for all such geographic 
units containing 20,000 or fewer people is changed to 000.
Telephone numbers 
Fax numbers
E-mail addresses
Social Security numbers 
Medical record numbers
Health plan beneficiary numbers 
Account numbers
All elements of dates (except year) for dates related to an individual, including: birth date, admission date, 
discharge date, date of death, all ages over 89 and all elements of dates (including year) indicative of such age, except 
that such ages and elements may be aggregated into a single category of age 90 or older
Certificate/license numbers
Vehicle identifiers and serial numbers, including license plate numbers 
Device identifiers and serial numbers
Web Universal Resource Locators (URLs) 
Internet Protocol (IP) address numbers
Biometric identifiers, including finger and voice prints
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Full face photographic images and any comparable images 
Any other unique identifying numbers, characteristics, or codes
5.
Assessment of Current Health Provider Relationship for HIPAA Consideration:
Does the Investigator or any member of the research team have a direct existing clinical relationship with any 
potential subject?
Yes, all subjects
Yes, some of the subjects 
No
If yes, describe the nature of this relationship.
Subjects with type 1 diabetes will be recruited from the surrounding area. Therefore it is 
possible that some patients may be under the care of the principle investigator, Dr 
Herzog, in the setting of the Yale Diabetes Center.
6.
Request for waiver of HIPAA authorization: (When requesting a waiver of HIPAA Authorization for either 
the entire study, or for recruitment purposes only. Note: if you are collecting PHI as part of a phone or email 
screen, you must request a HIPAA waiver for recruitment purposes.)
Choose one: For entire study: 
For recruitment purposes only: Yes 
X 
i.
Describe why it would be impracticable to obtain the subject’s authorization for use/disclosure of this 
data;
We are requesting a waiver of HIPPA Authorization in order to utilize the 
YNHH admission lists that would indicate patients we may not be able to 
contact prior to their discharge from the hospital. Such cases may be 
admissions that are over the weekends and holidays that may meet our 
criteria for inclusion. We would like to be able to identify Type 1 diabetic 
potential subjects that may be interested in participating, but were discharged 
prior to being able to discuss the study in person. We will contact the clinician 
that the patient is scheduled to see for follow up care and ask that they assist 
with referring the potential subject to contact us, or will allow us to speak to 
the subject at their next visit directly if the subject agrees when asked by their 
clinician.
ii.
If requesting a waiver of signed authorization, describe why it would be impracticable to obtain the 
subject’s signed authorization for use/disclosure of this data; We will obtain signed consent for the 
entire study if the subject agrees to participate.
By signing this protocol application, the investigator assures that the protected health information for 
which a Waiver of Authorization has been requested will not be reused or disclosed to any person or 
entity other than those listed in this application, except as required by law, for authorized oversight of 
this research study, or as specifically approved for use in another study by an IRB.
Researchers are reminded that unauthorized disclosures of PHI to individuals outside of the Yale HIPAA- 
Covered entity must be accounted for in the “accounting for disclosures log”, by subject name, purpose, date, 
recipients, and a description of information provided. Logs are to be forwarded to the Deputy HIPAA Privacy 
Officer.
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7.
Required HIPAA Authorization: If the research involves the creation, use or disclosure of protected health 
information (PHI), separate subject authorization is required under the HIPAA Privacy Rule. Indicate which of 
the following forms are being provided:
Compound Consent and Authorization form 
HIPAA Research Authorization Form
Consent Personnel: List the names of all members of the research team who will be obtaining 
consent/assent.
Raimund Herzog, M.D. ; Katrin Endrikat 
Yvette Strong; Domenico Trico, M.D.
8.
Process of Consent/Assent: Describe the setting and conditions under which consent/assent will be obtained, 
including parental permission or surrogate permission and the steps taken to ensure subjects’ independent 
decision-making.
The study will be described to the subject in detail, including the purpose and potential 
risks associated with the study. This will be explained by the physician, including the 
intervention and process for the study day. The subject will be required to read and sign 
the consent form approved by the HIC, and the patient will retain a copy of this consent 
to review. Additionally, the patient will sufficient time to ask questions during the 
screening and will be encouraged to contact investigators with further questions or 
concerns.
9.
Evaluation of Subject(s) Capacity to Provide Informed Consent/Assent: Indicate how the personnel 
obtaining consent will assess the potential subject’s ability and capacity to consent to the research being 
proposed.
In this study, we are not using vulnerable subjects. Subjects will be asked open-ended 
questions in order to evaluate their understanding of the research protocol and the risks 
involved.
10. Documentation of Consent/Assent: Specify the documents that will be used during the consent/assent process. 
Copies of all documents should be appended to the protocol, in the same format that they will be given to 
subjects.
A compound authorization and consent form will be used.
11. Non-English Speaking Subjects: Explain provisions in place to ensure comprehension for research involving 
non-English speaking subjects. Translated copies of all consent materials must be submitted for approval prior 
to use.
N/A
12. Consent Waiver: In certain circumstances, the HIC may grant a full or partial waiver of consent, 
depending on the study. If you will request either a waiver of consent, or a waiver of signed consent for this 
study, complete the appropriate section below.
Not Requesting a Consent Waiver 
Requesting a waiver of signed consent 
Requesting a full waiver of consent
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SECTION VIII: PROTECTION OF RESEARCH SUBJECTS
A. Waiver of signed consent: (Verbal consent from subjects will be obtained. If PHI is collected, 
information in this section must match Section VII, Question 6)
Requesting a waiver of signed consent for recruitment/screening only
Requesting a waiver of signed consent for the entire study (Note that an information sheet may be 
required.)
If requesting a waiver of signed consent, please address the following:
a.
Would the signed consent form be the only record linking the subject and the research?
Yes
No
b.
Does a breach of confidentiality constitute the principal risk to subjects? 
Yes
No
OR
c. Does the research pose greater than minimal risk?
granted.
No
Yes If you answered yes, stop. A waiver cannot be
AND
d. Does the research include any activities that would require signed consent in a non-research context? 
Yes
No
B. Full waiver of consent: (No consent from subjects will be obtained.)
Requesting a waiver of consent for recruitment/screening only
Requesting a full waiver of consent for the study (Note: If PHI is collected, information here must 
match Section VII, question 6.)
If requesting a full waiver of consent, please address the following:
a. Does the research pose greater than minimal risk to subjects?
waiver cannot be granted.
No
b. Will the waiver adversely affect subjects’ rights and welfare?
Yes If you answered yes, stop. A
Yes
No
c. Why would the research be impracticable to conduct without the waiver?
d. Where appropriate, how will pertinent information be returned to, or shared with subjects at a later date?
Confidentiality & Security of Data:
a. What protected health information (medical information along with the HIPAA identifiers) about subjects will be 
collected and used for the research?
Protected health information will include all blood work, urine pregnancy in women, 
medical history and physical examination results.
b.
How will the research data be collected, recorded and stored?
To comply with HIPAA standards, patient identification will be kept confidential. Subjects 
will be identified by code number, not by name during the data analysis. This data will be 
stored in computer files by code number, and their names will appear only on the initial 
form. Data that links the subject’s number to the subject will be kept in password
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SECTION IX: POTENTIAL BENEFITS
protected documents and kept on a secured server. Any data that contains patient’s 
identifying information will be released only with written consent from the patient.
c.
How will the digital data be stored?
CD
DVD
Flash Drive
Portable Hard
Drive  
 Secured Server
Laptop Computer
Desktop Computer
Other
d.
What methods and procedures will be used to safeguard the confidentiality and security of
the identifiable study data and the storage media indicated above during and after the subject’s participation in 
the study?
The documents will be password protected and held on a secured server which will only 
be available to the principle investigator and the co-investigators.
Do all portable devices contain encryption software? 
 Yes
No
If no, see http://hipaa.yale.edu/guidance/policy.html
e.
What will be done with the data when the research is completed? Are there plans to destroy the identifiable 
data? If yes, describe how, by whom and when identifiers will be destroyed. If no, describe how the data and/or 
identifiers will be secured.
Individual’s names will appear only on the initial forms and these will be kept under on a 
secured server. All data files that could link a code number to an individual study subject 
will be password protected. Once the study is completed zeroing will be used to remove 
any identifiable subject information.
f.
Who will have access to the protected health information (such as the research sponsor, the investigator, the 
research staff, all research monitors, FDA, QUACS, SSC, etc.)? (please distinguish between PHI and de-identified 
data)
Principle investigators and co-investigators only, no external agencies.
g. If appropriate, has a Certificate of Confidentiality been obtained?
N/A
h.
Are any of the study procedures likely to yield information subject to mandatory reporting   requirements?   
(e.g. HIV testing – reporting of communicable diseases; parent interview -incidents of child abuse, elderly abuse, 
etc.). Please verify to whom such instances will need to be reported.
N/A
Potential Benefits: Identify any benefits that may be reasonably expected to result from the research, either to the 
subject(s) or to society at large. (Payment of subjects is not considered a benefit in this context of the risk benefit 
assessment.)
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SECTION X: RESEARCH ALTERNATIVES AND ECONOMIC CONSIDERATIONS
Study subjects will derive no direct medical benefits from the study. However, the 
potential benefits of this study to society at large, and to the T1DM population in particular, 
are substantial in that the knowledge gained from this work should provide important 
information concerning the effects of recurrent, antecedent hypoglycemia on CNS 
responses to hypoglycemia. This information could contribute significantly to clinical 
indications and patient selection for the islet transplantation procedure.
1.
Alternatives: What other alternatives are available to the study subjects outside of the research?
No alternative treatments are available.
2.
Payments for Participation (Economic Considerations): Describe any payments that will be made to 
subjects, the amount and schedule of payments, and the conditions for receiving this compensation.
Subjects assigned to Cohort 1 will receive a total of $350 for participating in the study 
($50 for the completion of the first scan and $300 for completion of the second scan). 
Subjects assigned to Cohort 2,3 and 5 will receive $300 for completing the study. 
Subjects assigned to Cohort 4 will be paid $60 for their participation. Payments will be 
given to the subject via a Bank of America pre-paid debit card using the Yale electronic 
payment service. 
3.
Costs for Participation (Economic Considerations): Clearly describe the subject’s costs associated with 
participation in the research, and the interventions or procedures of the study that will be provided at no cost 
to subjects.
Subjects will not be responsible for any costs associated with the study or the screening 
portion prior to the study, including blood work, history and physical examination, or urine 
pregnancy test. We believe that given the length and number of procedures performed, 
the remuneration is fair and is not an excessive inducement for people to volunteer for 
the study.
4.
In Case of Injury: This section is required for any research involving more than minimal risk.
a.
Will medical treatment be available if research-related injury occurs?
b.
Where and from whom may treatment be obtained?
c.
Are there any limits to the treatment being provided?
d.
Who will pay for this treatment?
e.
How will the medical treatment be accessed by subjects?
If injury were to occur, we will provide any care of treatment required. Our study has no 
provision for additional compensation. While participating in the study procedures the 
overseeing physician, Dr. Herzog or one of his designated co-investigators will be 
responsible for providing treatment. If necessary and the treatment requirement exceeds 
what can be provided at the study site, the participant will be transferred to the YNHH 
Emergency Department for further care. The participant’s insurance will be billed for the 
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cost of this treatment. Should problems arise after completion of the study the subjects 
will be provided with a telephone number to reach the PI’s office and additional numbers
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that will allow 24hr access to the study investigators.
TIMELINE and Blood Drawing Schedule for Hypoglycemic clamp study and recovery
Time
Real 
Time
YSI
glucose 
q5 min
Insulin
Glucagon
Catachols
GCMS /
other 
analytes
Lactate & 
Ketones
Cortisol 
and GH
Notes
A.1. 
Baseline
X
X
X
X
X
X
B.1. 
Start 
insulin/ 
start 
clock
Glucose is 
checked 
every 5 
minutes
X
X
X
Enter 
scanner
B.2. 
B.1 +30
X
X
X
X
B.3. 
B.1 +60
X
X
X
X
B.4. 
B.1 +90
X
X
X
X
C.1.
 
BHB
Baseline
Start 
BHB
infusion
Glucose is 
checked q5 
min
X
X
X
X
X
X
Target 
glucose
~55mg/dl
C.2. 
C.1 +5
↓
X
↓
C.3. 
C.1 +10
↓
X
↓
C.4. 
C.1 +15
↓
X
↓
C.5. 
C.1 +30
↓
X
X
X
X
X
↓
C.6. 
C.1 +60
↓
X
X
X
X
X
X
↓
C.7. 
C.1 +90
↓
X
X
X
X
X
↓
C.8. 
C.1 +120
Glucose 
check
X
X
X
X
X
X
End Clamp
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COHORT 1:
Schedule of Events:
Screening 
Visit 
Session A
Proton-NMR 
Spectroscopy 
Session B
Carbon 13-NMR 
Spectroscopy 
Session C
Total Blood 
Collected
Informed Consent
X
Medical History
X
Physical Exam
X
EKG
X
Hematocrit (3 mLs)
X (3mLs)
Creatinine (2 mLs)
X (2mLs)
Liver Function Tests (3mLs)
X (3mLs)
HbA1c (3mLs)
X (3mLs)
Inclusion/Exclusion Check list
X
Record Blood Sugar Levels 5x 
daily *
X
Complete questionnaire for glucose 
readings <54mg/dl *
X
Schedule two MR sessions
X
Urine Pregnancy (females)
X
X
IV Placement
X
Hypoglycemic Clamp
X
Blood Volume per visit
11 ml
none
< 250ml
< 250ml
*: For diabetic subjects only
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COHORT 2:
Schedule of Events:
Screening 
Visit 
Session A
Carbon 13-NMR 
Spectroscopy 
Session C
Total Blood 
Collected
Informed Consent
X
Medical History
X
Physical Exam
X
EKG
X
Hematocrit (3 mLs)
X (3mLs)
Creatinine (2 mLs)
X (2mLs)
Liver Function Tests (3mLs)
X (3mLs)
HbA1c (3mLs)
X (3mLs)
Inclusion/Exclusion Check list
X
Record Blood Sugar Levels 5x 
daily *
X
Complete questionnaire for glucose 
readings <54mg/dl *
X
Schedule MR sessions
X
Urine Pregnancy (females)
X
X
IV Placement
X
Hypoglycemic Clamp
X
Blood Volume per visit
11 ml
< 250ml
< 250 ml
*: For diabetic subjects only
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COHORT 3:
Schedule of Events:
Screening 
Visit 
Session A
Proton-NMR 
Spectroscopy 
Session B
Total Blood 
Collected
Informed Consent
X
Medical History
X
Physical Exam
X
EKG
X
Hematocrit (3 mLs)
X (3mLs)
Creatinine (2 mLs)
X (2mLs)
Liver Function Tests (3mLs)
X (3mLs)
HbA1c (3mLs)
X (3mLs)
Inclusion/Exclusion Check list
X
Record Blood Sugar Levels 5x 
daily *
X
Complete questionnaire for glucose 
readings <54mg/dl *
X
Schedule MR sessions
X
Urine Pregnancy (females)
X
X
IV Placement
X
Hypoglycemic Clamp
X
Blood Volume per visit
11 ml
< 250ml
< 250 ml
*: For diabetic subjects only
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COHORT 4:
Schedule of Events:
Screening 
Visit 
Session A
Proton-NMR 
Spectroscopy 
Session B
Informed Consent
X
Inclusion/Exclusion 
Check list
X
Urine Pregnancy 
(females)
X
X
Blood Volume per visit
none
none
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COHORT 5:
Schedule of Events:
Screening 
Visit 
Session A
Cognitive 
Testing 
Session D
Total Blood 
Collected
Informed Consent
X
Medical History
X
Physical Exam
X
EKG
X
Hematocrit (3 mLs)
X (3mLs)
Creatinine (2 mLs)
X (2mLs)
Liver Function Tests (3mLs)
X (3mLs)
HbA1c (3mLs)
X (3mLs)
Inclusion/Exclusion Check list
X
Record Blood Sugar Levels 5x 
daily *
X
Complete questionnaire for glucose 
readings <54mg/dl *
X
Schedule cognitive testing session
X
Urine Pregnancy (females)
X
X
IV Placement
X
Hypoglycemic Clamp
X
Alternate Fuel (BHB) infusion
X
Cognitive Testing
X
X
Blood Volume per visit
11 ml
< 250ml
< 250 ml
*: For diabetic subjects only
Any unused serum collected from all cohorts may be kept for additional metabolic studies or for 
confirmation of findings at a future time for both subject groups (non-diabetic and diabetic).
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