Clinical trial NCT01232946
Conditions: Type 2 Diabetes
Keywords: Diabetes Myocardial metabolism
Sponsor: Indiana University
Start date: 2012-01-01; Completion date: 2017-09-08
Enrollment: 30 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
27 Type 2 diabetic subjects (HbA1c 7.5 - 9.5%) currently treated with diet and exercise alone or with oral antidiabetic agents will be randomly assigned to one of three treatment groups: insulin detemir, liraglutide, or liraglutide plus detemir (9 subjects per group), on a background of metformin 2000 mg per day. After 3 months' treatment, PET measurements of myocardial fuel selection will take place, under fasting conditions in the morning following that day's treatment injection(s).

Detailed description:
The investigators will test the hypothesis that effects of liraglutide plus insulin detemir on myocardial fuel selection will be greater than the effects of either agent alone. 27 Type 2 diabetic subjects (HbA1c 7.5 - 9.5%) currently treated with diet and exercise alone or with oral antidiabetic agents will be randomly assigned to one of three treatment groups: insulin detemir, liraglutide, or liraglutide plus detemir (9 subjects per group). All subjects will undergo an initial standardization of background treatment to metformin 2000 mg per day, followed by randomized assignment to 3 months' treatment with liraglutide 1.8 mg/day (once daily each morning, tapering up according to label instructions) and/or detemir (administered twice daily, initially at 10 units per day then titrated to achieve fasting morning glucose readings below 130 mg/dL). PET measurements of myocardial fuel selection will take place at the end of this treatment period, under fasting conditions in the morning following that day's treatment injection(s).

Arms: arm 1: Type 2 diabetic subjects will be assigned to 3 months of treatment with 1.8mg liraglutide administered once daily in addition to background metformin 2000mg/day. PET measurements of myocardial glucose uptake will take place at the end of treatment. arm 2: Type 2 diabetic subjects will be assigned to 3 months of treatment with insulin detemir administered twice daily in addition to background metformin 2000mg/day. PET measurements of myocardial glucose uptake will take place at the end of treatment. arm 3: Type 2 diabetic subjects will be assigned to 3 months of treatment with a combination of liraglutide and insulin detemir in addition to background metformin 2000mg/day. PET measurements of myocardial glucose uptake will take place at the end of treatment.
Interventions: intervention 1: liraglutide intervention 2: insulin detemir intervention 3: liraglutide plus insulin detemir
Intervention descriptions: intervention 1: 1.8mg subcutaneous qd for 3 months intervention 2: 5units subcutaneous bid titrated to fasting glucose of \<130mg/dL for 3 months intervention 3: liraglutide 1.8mg subq qd plus insulin detemir 5units bid titrated to fasting blood glucose \<130mg/dL for 3 months.

Protocol documents:
Prot_SAP_000.pdf: 
 
1 
Cooperation of Insulin and GLP-1 on Myocardial Glucose Uptake 
NCT01232946 
 
Principal Investigator 
Kieren J Mather MD FRCPC 
1120 West Michigan Street, CL365 
Indianapolis IN 46202 
317-278-7826 T 
317-274-2695 F 
kmather@iu.edu 
 
Investigator-initiated project funded by Novo Nordisk 
 
Final protocol as implemented 
 
Document Date: 10/29/2010 


 
2 
Coop 
A. Study Aim 
Extrapancreatic effects of glucagon-like peptide-1 (GLP-1) present novel therapeutic opportunities. In animal 
studies, and in our preliminary data in humans, GLP-1 can modulate cardiac metabolism by augmenting use of 
glucose for fuel. Furthermore, animal studies have demonstrated synergistic actions of insulin and GLP-1 to 
augment myocardial glucose uptake, with net uptake from combined exposure greater than that achieved by 
either agent individually. This suggests that therapeutic interventions using combinations of insulin and GLP-1 
agonism may have important advantages in terms of effects on myocardial fuel metabolism, perhaps providing 
protection against consequences of diabetes-related changes in myocardial metabolism in ischemia or 
contractile dysfunction. 
Liraglutide is a GLP-1 agonist that has recently been approved in the United States and in Europe for clinical 
use in the treatment of type 2 diabetes mellitus. There is emerging evidence from animal studies that liraglutide 
may provide protection against myocardial ischemia, but to our knowledge effects of liraglutide on myocardial 
glucose uptake, alone or in combination with insulin, have not been explicitly evaluated in any animal model or 
in humans. Here we propose physiologic studies of the effect of liraglutide to magnify insulin-stimulated 
myocardial glucose uptake in humans with Type 2 diabetes mellitus, overcoming myocardial insulin resistance 
and maximizing the shift to carbohydrate fuel utilization. 
We will test the hypothesis that effects of liraglutide plus insulin detemir on myocardial fuel selection will be 
greater than the effects of either agent alone. 27 Type 2 diabetic subjects (HbA1c 7.5 – 9.5%) currently treated 
with diet and exercise alone or with oral antidiabetic agents will be randomly assigned to one of three treatment 
groups: insulin detemir, liraglutide, or liraglutide plus detemir (9 subjects per group). All subjects will undergo 
an initial standardization of background treatment to metformin 2000 mg per day, followed by randomized 
assignment to 3 months’ treatment with liraglutide 1.8 mg/day (once daily each morning, tapering up according 
to label instructions) and/or detemir (administered twice daily, initially at 10 units per day then titrated to 
achieve fasting morning glucose readings below 130 mg/dL). PET measurements of myocardial fuel selection 
will take place at the end of this treatment period, under fasting conditions in the morning following that day’s 
treatment injection(s). The primary study endpoint will be myocardial glucose uptake. This and other metabolic 
and hemodynamic endpoints measured at steady state of each study condition will be compared across 
treatment conditions to allow evaluation of additive actions of the combination exposure compared to actions of 
liraglutide or insulin detemir alone.  
The results from these studies will allow objective evaluation of the effects of combining liraglutide with insulin 
detemir on myocardial fuel selection in humans with type 2 diabetes. These data will provide a rationale for 
further studies of treatments with liraglutide (with or without insulin) in the setting of myocardial metabolic 
dysfunction. 


 
3 
B. Significance and Background 
B1. Significance 
Cardiac disease is currently the second most common cause of mortality in North America, and the obesity 
epidemic is projected to further increase heart disease incidence and prevalence (1, 2). Current treatments to 
reduce atherosclerotic heart disease by control of cholesterol, blood pressure, and anti-thrombotics are clearly 
of benefit, and have contributed to important reductions in heart disease mortality. However, even with the best 
current treatments, elevated rates of heart disease in obesity and diabetes persist (3, 4). Although it is 
worthwhile to pursue improvements in therapeutic approaches against these established targets, there is also 
clear value in pursuing new therapeutic targets. 
One emerging target is therapeutic modulation of myocardial metabolism, specifically modulation of fuel 
selection (5). Control of fuel selection under conditions of ischemia has clear therapeutic potential and is the 
main focus of the current investigations. More generally there is an emerging understanding of the 
pathophysiologic connections between abnormalities in cardiac fuel metabolism and abnormalities in cardiac 
function. Therefore therapies that modulate fuel selection promise to be beneficial in heart disease more 
broadly, including in patients with chronic angina, congestive heart failure, and diabetic cardiomyopathy. GLP-1 
based treatments are particularly promising in targeting myocardial fuel selection because 1) the magnitude of 
induced changes is large; and 2) these treatments represent a known quantity in terms of toxicity and 
tolerability, so efforts in pursuit of this therapeutic application are unlikely to be derailed by unexpected 
toxicities late in development.  
Under ischemia, it is conceptually preferable to metabolize glucose over other fuel sources because 
carbohydrates require fewer moles of oxygen required per mole of ATP derived compared to fats.  Also, driving 
glucose uptake despite stress-induced elevation in circulating free fatty acids helps ensure that carbohydrates 
are available for anaerobic metabolism, should that be required. Insulin and GLP-1 each help drive myocardial 
glucose uptake, and this is the presumed mechanism underlying the cardioprotective effect of GLP-1 that has 
been observed in preclinical studies (presented in detail below). Insulin is difficult to apply therapeutically with 
the goal of improving myocardial glucose uptake in the setting of myocardial infarction. Two main features are 
limiting: First, sensitivity to insulin action for systemic glucose disposal varies widely in the population, and is 
subject to acute changes with physiologic stress. This means that it is difficult to pre-specify a minimum dose 
that is sufficient to drive myocardial glucose uptake in all individuals. Second, in a given individual the 
therapeutic window of insulin is fairly narrow. This means that it is difficult to pre-specify a maximum dose that 
is guaranteed not to produce hypoglycemia in all individuals. This combination of problems means that the 
correct application of insulin simply cannot be pre-specified, but rather it requires a period of dose adjustment 
and ongoing close monitoring to ensure that systemic glucose concentrations are maintained within the target 
range. But even this approach does not address the true goal of driving myocardial glucose uptake; an 
adequate effect is simply presumed to take place in response to the systemic hyperinsulinemia. The use of 


 
4 
GLP-1 – based treatments instead of insulin, or in combination with insulin, presents an opportunity to 
maximize the beneficial effects to drive carbohydrate fuel selection and concurrently minimize the risks of 
treatment-induced hypoglycemia. 
B2. Background 
We propose studies that will systematically evaluate changes in myocardial fuel selection induced by the GLP-
1 agonist liraglutide, alone and in combination with exogenous hyperinsulinemia, in vivo in humans. These 
studies are predicated on the following key points, briefly summarized in the following text. 
1) GLP-1 acts on the heart to drive selection of glucose as a fuel 
2) Modulation of myocardial fuel selection is a promising therapeutic target 
3) Clinically approved liraglutide dosing is likely to have effects on myocardial fuel selection 
B2.1. GLP-1 acts on the heart to drive selection of glucose as a fuel 
Molecular Biology of Glucagon-Like Peptide 1 GLP-1 is produced primarily in a subset of enteroendocrine 
cells (L-cells) that are distributed in the distal small bowel and colon. It is produced from proglucagon, which is 
cleaved by prohormone convertase-1 into glicentin, GLP-1, GLP-2, and oxyntomodulin (6). There are 2 
equipotent naturally occurring active forms of GLP-1, namely GLP-1[7-37] and GLP-1[7-36]amide (7). Both 
forms are rapidly degraded by circulating dipeptidylpeptidase 4 (DPP-4), and the resulting fragments are 
cleared by the kidney (6).  The GLP-1 receptor is a seven transmembrane segment G-protein coupled receptor 
that signals via cyclic AMP (8), with wide tissue distribution including expression in the heart and CNS (9, 10). 
This receptor mediates GLP-1 actions in the pancreas (6, 11). Direct GLP-1 actions on the heart appear to be 
mediated by cAMP (8) and therefore by this receptor, but evidence also implicates involvement of a putative 
alternative receptor (12-14). The best-recognized actions of GLP-1 are in the pancreatic islets, where together 
with GIP it is responsible for amplification of glucose-stimulated insulin release (6). This effect is produced by 
actions in beta cells (amplifying the intracellular mechanisms linking glucose sensing to insulin secretion) and 
by actions in alpha cells (suppressing the production of glucagon) (15). Effects of GLP-1 on whole-body 
metabolism are exerted principally via these islet-derived hormones (6, 15).   
Effects of GLP-1 on Cardiac Glucose Metabolism   GLP-1 effects on myocardial function and fuel selection 
have been demonstrated with in vitro studies, with studies of isolated hearts, and with in vivo animal studies.   
Cardiomyocytes GLP-1 receptors are expressed in the heart (9, 10). In isolated rat cardiomyocytes GLP-1 
(10-8 mol/L) increased intracellular cAMP, implicating signaling via the classical GLP-1 receptor (8). In skeletal 
muscle strips and cultured myotubes, GLP-1 directly stimulated glucose uptake and subsequent steps of 
intermediary metabolism (ED50 ~ 10-11 mol/L), independent of and additive to insulin (16).   
Isolated hearts Effects of GLP-1 on isolated perfused rat hearts have been studied using the Langendorff 
preparation. Importantly, effects observed with this preparation are independent of systemic hormonal changes 
and neurologic inputs that may contribute to actions observed in vivo. GLP-1 (500 pmol/L) acutely and 
significantly increased glucose consumption by the isolated heart from ~33 to ~81 μmol/min/g (i.e. ~2.5-fold) 


 
5 
(17). Insulin and GLP-1 both increased glucose uptake via changes in glucose transporters, but the GLP-1 
effect was independent of the classical insulin signaling cascade (18). These observations and other in vitro 
data also suggest separate and additive effects of insulin and GLP-1 on myocardial glucose uptake (19). 
These data demonstrate direct effects of GLP-1 on the myocardium to shift fuel selection toward glucose and 
away from fatty acids, analogous to but independent of actions of insulin.   
Isolated rat hearts have also been used to study the effects of GLP-1 on ischemia/reperfusion injury (18, 20-
23). GLP-1 perfusion at the time of injury enhanced recovery of contractile function, reduced post-ischemia 
functional deficit, and reduced infarct size. The GLP-1 receptor antagonist Exendin[9-39]  blocked these 
effects,  suggesting the effects were mediated at least in part via the  classical GLP-1 receptor (21).  These 
observations were confirmed in studies using other GLP-1 agonists including the GLP-1 fragment GLP-1[9-36] 
(22).  These data suggest that GLP-1 may be therapeutically beneficial in ischemia by acting to drive 
myocardial glucose utilization.   
 In vivo –Animals Studies of ischemia and pacing-induced dilated cardiomyopathy support the potential for 
therapeutic  benefit from GLP-1 – based treatments in vivo under pathophysiologic conditions. The key studies 
are summarized in Table B1. Briefly, in 3 different mammalian models of heart disease, native GLP-1 or GLP-
1 agonists provided protection against ischemia-induced cardiac damage, or improved myocardial contractile 
function (12, 17, 20-22, 24, 26, 29). These beneficial effects were related to actions of the agonists to augment 
myocardial glucose uptake, which was increased by 1.5 to 3 fold above baseline (e.g. from 5.4 to 7.9 μmol/min 
in (12) and from ~4 to ~12 μmol/min in (29), both p<0.05).   
These studies demonstrate that GLP-1 can modulate myocardial fuel selection in vivo. The beneficial effects 
against ischemia were related to effects on myocardial glucose uptake.  These effects of GLP-1 on myocardial 
metabolism may not require insulin since the variant peptide, GLP-1[9-36] was able to recapitulate the actions 
of native GLP-1[7-36] including augmentation of myocardial insulin sensitivity but specifically without 
Reference  
Animal  Agonist:Dose  
Model:Outcome 
Kavianipour  (24)   
Pig 
GLP-1:  3 pmol/kg/min  for 5 
hrs   
Acute ischemia: Reduced  intramyocardial pyruvate and  lactate 
accumulation under acute  ligation ischemia   
Nikolaidis  (25)   
Dog 
 
 
GLP-1:  1.5  pmol/kg/min 
for  48 hrs   
Pacing-induced cardiomyopathy:  Improved contractile function, 
augmented myocardial glucose  uptake, reduced fatty acid uptake. 
Nikolaidis  (12)   
Dog 
GLP-1, GLP-  1[9-
36]:  1.5  pmol/kg/min for  48 
hrs   
Pacing-induced cardiomyopathy:  Both agonists improved 
contractile  function, augmented basal and  insulin-stimulated 
myocardial  glucose uptake. 
Bose  (20, 21)   
Mouse 
GLP-1:  4.8  pmol/kg/min   
Ligation ischemia/reperfusion  model: Reduced infarct 
size,  blocked by GLP-1 receptor  antagonism 
Dokken (26) 
Mouse 
GLP-1:  30 pmol/kg/min  for 2 
hrs   
Ligation infarct: Postconditioning  approach, reduced infarct size 
Sonne (22) 
Mouse 
Liraglutide:  200 μg/kg/d for  7 
days   
Ligation infarct: Improved survival,  reduced infarct size, reduced 
rate  of myocardial rupture 
Noyan-  Ashraf 
(17) 
Mouse 
Liraglutide:  200 μg/kg/d for  7 
days   
Ligation infarct: Improved survival,  reduced infarct size, reduced 
rate  of myocardial rupture   
Kristensen (27) 
Pig 
Liraglutide 10  μg/kg/d for 3 
days   
Ligation infarct: No reduction in survival, modest nonsignificant 
reduction in infarct size. 
Timmers (28) 
Pig 
Exenatide 10  μg twice daily 
for 3 days  
Ligation infarct: Reduced infarct size, preserved contractile function 
Table B1. Animal studies of in vivo actions of GLP-1 agonists on the heart 


 
6 
engendering an insulinotropic response (12, 30). As noted above, in vitro observations suggest that actions of 
insulin and GLP-1 to augment glucose uptake are distinct and additive (18, 19). In the dog studies, separate 
and combined infusions of insulin and GLP-1 were evaluated (12, 29). With combined exposure myocardial 
glucose uptake was markedly enhanced (~9-fold above baseline), greater than either agent alone and greater 
than the simple addition of the individual responses. We propose to undertake studies to specifically explore 
these effects in humans, using Liraglutide as the GLP-1 agonist. 
In vivo – Humans A small number of preliminary clinical studies have been undertaken using GLP-1 to treat 
heart disease in humans. In one small non-randomized study of 6 diabetic patients with congestive heart 
failure, GLP-1 at 4.0 pmol/kg/min for 3 days resulted in a nonsignificant trend toward improved contractile 
function (31). Three proof-of-principle human studies focusing on effects of GLP- 1 on contractile function have 
been reported (29, 32, 33). These were non-randomized studies of effects of GLP-1 infusions on functional 
outcomes in patients with cardiac dysfunction. GLP-1 exposures ranged from 1.25 to 2.5 pmol/kg/min for 72 
hours to 14 days, differing by study. Overall, significant improvements were noted in multiple 
echocardiographic measures of contractile function (ejection fraction, global and regional wall motion scores), 
functional status (distance on a 6 minute walk test and VO2max), and quality of life indices. Cardiac glucose 
metabolism was not assessed in any of these studies. Although these are not rigorously controlled clinical 
trials, they do support the potential for beneficial effects of GLP-1 in heart disease in humans. No human 
studies of anti-ischemic effects of GLP—based treatments have been reported, and no rigorous studies of the 
physiologic effects of GLP-1 itself, DPP4 inhibitors, or GLP-1 agonists such as liraglutide on myocardial 
metabolism in humans have been reported.   
B2.2. Modulation of myocardial fuel selection is a promising therapeutic target 
Therapeutic modulation of myocardial fuel selection is an increasingly active area of investigation, with 
burgeoning interest based on the clear potential for development of novel therapeutic interventions (5, 34-37).  
Perhaps the most classical example of this treatment approach is the application of insulin at the time of 
infarction. Combined infusions of glucose, insulin, and potassium in the setting of myocardial infarction have 
reduced mortality in many studies of subjects with and without diabetes (38-41). However, these benefits have 
not been seen other large studies (42-44), perhaps reflecting a particular benefit among diabetic patients from 
modulation of fuel selection (45). Other emerging approaches to modulation of fuel selection include inhibition 
of fatty acid metabolism (using e.g. perhexilene, a carnitine palmitoyl tranferase inhibitor, or trimetazidine, a 
partial fatty acid oxidation inhibitor). Early clinical studies suggest that these agents are effective treatments for 
ischemia manifesting as angina (34, 46-48), but alternatives with less toxicity or improved efficacy are needed. 
GLP-1 agonism with Liraglutide may present a valuable alternative approach, with good effectiveness and 
minimal toxicity.  


 
7 
B2.3. Liraglutide: Adequacy of Exposure and Side Effects 
The affinity of GLP-1 for the classical GLP-1 receptor is on the order of 10-10 mol/L (49, 50). The active moiety 
of liraglutide is a native GLP-1 (7-36) fragment, and despite the modifications that produce a prolonged half-life 
this agent has similar GLP-1 receptor affinity binding characteristics on classical GLP-1 receptors (51, 52). The 
circulating concentrations of liraglutide achieved with standard clinical dosing are in the micromolar range 
following a single subcutaneous injection (53), suggesting that the GLP-1 receptors are fully stimulated for the 
majority of the duration of exposure. This exceeds the exposure resulting from the physiologic GLP-1 response 
to meals in humans, which produces GLP-1 concentrations at the low end of the receptor’s affinity range(13, 
54, 55). Myocardial metabolism is significantly modified with intravenous infusions of native GLP-1 achieving 
circulating concentrations of ~160 pmol/L (12, 29, 56-58). It is therefore reasonable to predict that clinically 
used doses of liraglutide will be sufficient to engender GLP-1 mimetic effects on myocardial fuel selection. 
Common side effects seen with liraglutide include headache, nausea, diarrhea, respiratory tract infections, and 
anti-liraglutide antibody formation (59). Between 5 and 10% of patients find the side effects intolerable and 
withdraw from treatment. GI symptoms are by far the most common, and are dose-related. Up to 41% of 
patients reported such symptoms in the five major FDA-registration trials (versus 17% of trial subjects treated 
with comparator agents). These effects are generally transient, lasting only days, and overall are well tolerated 
(at least 90% of patients continue with treatment). Hypoglycemia is a rare event when native GLP-1 is given 
alone, and GLP-1 is well tolerated under fasting conditions (57); parallel observations have been made for 
liraglutide where hypoglycemia rates in liraglutide-treated subjects during the FDA registration trials were 2.6 
cases per 1000 patient-years, representing 7 subjects in the entire trial experience (59). Six of these subjects 
were co-treated with a sulfonylurea type insulin secretagogue. Other rare but important adverse effects include 
hypersensitivity/urticaria (occurring in 0.8%, versus 0.4% of comparator-treated subjects); pancreatitis (seen in 
7 liraglutide-treated subjects versus 1 comparator-treated subject, representing 2.2 vs 0.6 cases per 1000 
patient-years); and papillary thyroid carcinoma (6 versus 1 cases, 1.9 versus 0.6 cases per 1000 patient-
years). Due principally to data from preclinical development testing of Liraglutide there is a concern about 
hyperplasia of thyroid calcitonin-secreting (C)-cells potentially leading to medullary carcinoma of the thyroid 
(MTC) which was observed in rodents. The FDA labeling for Liraglutide contains a boxed warning about this 
issue, stating that 'it is unknown whether liraglutide causes C-cell tumors in humans, as human relevance 
could not be demonstrated in clinical or nonclinical studies'  (59). Liraglutide is contraindicated for use in 
patients with genetic predisposition to MTC, such as a personal or family history of medullary thyroid 
carcinoma or Multiple Endocrine Neoplasia syndrome Type 2  (59) . 
B3. Innovation 
There is intense interest in extrapancreatic actions of GLP-1, including in particular effects of GLP-1 on the 
heart (12, 17, 18, 21-25, 31-33, 60-74). Effects of GLP-1 to modulate myocardial fuel selection have been 
clearly established in preclinical work (presented in detail below) and this promises to result in truly novel 
applications of GLP-1 – based treatments for heart disease (61, 75).  Liraglutide is one such treatment, and 


 
8 
having undergone clinical development testing for use in treating diabetes, the toxicities and averse event 
profile are already well understood. The studies proposed here are the first to systematically evaluate the 
physiologic interactions of liraglutide and insulin in the regulation of myocardial fuel selection in humans, and 
will be of direct relevance to the clinical application of liraglutide as a 
therapeutic modulator of myocardial fuel selection. 
C. Preliminary Data 
Insulin and Myocardial Fuel Selection: We have recently completed 
a set of studies using PET to demonstrating the effects of insulin on 
myocardial substrate selection. We 
made PET measurements on 2 
consecutive mornings. Day 1 
measurements were made under 
fasting conditions (i.e. fatty acid-
dependent) and the Day 2 
measurements were made under 
steady state conditions using a 
hyperinsulinemic euglycemic 
clamp (i.e. carbohydrate-
dependent conditions; insulin 
exposure 120 mU/m2/min). We 
studied 9 lean males (age 
[mean±SD] 34.1±9.0 years; body mass index 22.6±2.4 kg/m2, 17.5±6.6 
percent fat) and 8 obese type 2 diabetic males (age 39.1±9.2 years; body 
mass index 34.2±7.3 kg/m2*, 37.6±13.2 percent fat* (*p<0.05 vs. lean 
subjects). Myocardial free fatty acid metabolism was measured using a 
proprietary tracer (18F-thiapalmitate, FTP; in the current proposal we will 
use a standard 11C-palmitate fatty acid tracer). In parallel with the current 
proposal, we also measured total myocardial oxidative metabolism, and 
myocardial perfusion using 11C-acetate. 
In order to minimize group differences in fuel availability on the non-insulin 
clamp study day, glycemic management in diabetic subjects was 
standardized by the PI prior to the PET measurements. Diabetic subjects 
(DM2) underwent intensification of their diabetes management using 
basal/bolus insulin for 4 weeks prior to these studies. With this 
intervention, fasting glucose levels achieved with basal insulin from the 
preceding night were approximately euglycemic (lean [mean+SD] 90.0±6.9 
 
Figure C2. Effects of insulin on 
myocardial oxygen consumption 
and blood flow (by PET), and 
systemic hemodynamics (by 
impedance cardiography). HR, 
h
t
t
MAP
t
i l
 
Figure C1. Effects of insulin on fatty acid 
kinetics measured using 18F-thiapalmitate. 
FFA=free fatty acids; FAO=fatty acid 
oxidation rate. 


 
9 
vs DM2 112.7±19.2 mg/dL, p=NS comparing groups). Insulin suppression of circulating fatty acids with the 
insulin/glucose clamp was overall comparable between groups (Figure C1: lean from 523±134 to 21±4; DM2 
from 528±149 to 85±8 µmol/L, p<0.001 for insulin effect and p=NS for group by insulin interaction). However, 
at steady state insulin infusion the ambient free fatty acid concentrations were significantly different (p=0.001; 
Figure C1 upper panel inset). Insulin induced a marked shift in myocardial fuel selection (Figure C1), with a 
>70% reduction in absolute rates of fatty acid oxidation (p=0.02). Comparable magnitudes of the insulin-
induced shift in kinetics of fatty acids were seen in the diabetic subjects compared to the lean subjects. 
However, the residual difference in fatty acid availability contributed to statistically significant differences in 
fatty acid uptake and oxidation between diabetic and control subjects under steady state insulin exposure 
(Figure C1 inset). Therefore the diabetic subjects exhibited 
net resistance to insulin’s effects to shift myocardial fuel 
selection. This was accompanied by resistance to insulin’s 
effects on blood pressure and myocardial blood flow as well 
(Figure C2). 
These data demonstrate impairments in insulin’s effects on 
myocardial fuel selection in Type 2 diabetes, and 
demonstrate impairments in the hemodynamic responses to 
insulin in this population. They also demonstrate our technical 
capacity to undertake studies the studies proposed. In 
secondary analyses under the current proposal we will 
evaluate whether liraglutide can help overcome these defects 
in insulin action in Type 2 diabetic subjects. 
GLP-1 and Myocardial Fuel Selection: In contrast to the 
effects of insulin on the heart, very little is known about the 
effects of GLP-1 on the heart in humans. Under a protocol 
recently funded by the NIH, we have so far evaluated these 
effects of native GLP-1 in 4 healthy lean non-diabetic male 
subjects (age 32±12 yrs, body mass index 23.9±1.6 kg/m2). 
Subjects underwent PET measurements of rates of 
myocardial glucose uptake, blood flow, and total oxidation 
under control conditions, and then again the next day 
following a 10 hour exposure to GLP-1 (1.5 pmol/kg/min). 
Representative images demonstrating the effect of GLP-1 
on myocardial glucose uptake are presented in Figure C3. 
The quantitative findings for all 4 subjects are presented in Figure C4. Kinetic analyses were performed using 
modeling as currently proposed (details below). Similar to the effect magnitude reported in preclinical studies, 
exposure to GLP-1 (1.5 pmol/kg/min) resulted in an average 2.1-fold increase in myocardial glucose uptake 
 
Figure C3 FDG PET images of myocardial 
glucose uptake under control fasting conditions 
and following overnight infusion of GLP-1. 
Images are presented with comparable color 
gradients. 
 
Figure C4 Effects of GLP-1 on myocardial glucose 
uptake, perfusion, and total oxidation rate. N=4 


 
10
(from 11.9±8.7 mean±SD to 25.7±15.6 μmol/100g/min). In this pilot dataset this change is already statistically 
significant (p=0.04), clearly demonstrating the sensitivity of this testing methodology to the anticipated effect 
sizes. 
A concurrent nonsignificant increase in 
myocardial blood flow was seen, accompanied 
by a significant increase in myocardial oxygen 
consumption (acetate disappearance, 
expressed as MvO2; Figure C4). Significant 
chronotropic or hemodynamic effects of GLP-1 
were not evident with this small sample size. (Table C1).   
We anticipate that liraglutide will exert similar effects on myocardial fuel selection. Our principal question is 
whether the effect of combined exposure is greater than the effect of liraglutide alone. We have no data to date 
evaluating this question. 
 D. Approach 
D1. Protocol Overview  
We will test the hypothesis that effects of liraglutide plus insulin detemir on 
myocardial fuel selection will be greater than the effects of either agent alone. We expect to demonstrate that 
liraglutide and insulin detemir can act in an additive way to affect myocardial fuel selection. We will study 
subjects exposed to each agent alone and in combination.  
PET will be used to measure myocardial perfusion and rates of total oxidation, 
fatty acid uptake, fatty acid oxidation and glucose uptake. Full details are 
provided in Section D5 below; in brief, subjects will be randomized at enrollment 
to one of three treatments (insulin detemir alone, liraglutide alone, or combined 
insulin detemir and liraglutide). Each subject will be studied once, following a 3 
month treatment with the assigned agent(s).  
D2. Rationale Both GLP-1 and insulin exert actions to modulate myocardial fuel 
selection. The combination of insulin and the GLP-1 agonist liraglutide may be 
advantageous in maximizing the capacity to therapeutically modulate myocardial 
glucose uptake, for example in circumstances of acute myocardial ischemia.  
Molecular and tissue studies indicate that insulin and GLP-1 act via separate 
signaling pathways to exert additive effects on glucose transport (16, 65). These 
observations plus important physiologic differences between the systemic actions 
of these agents suggest that their effects on fuel selection may arise from distinct 
and additive mechanisms.  
D3. Anticipated Results & Interpretations  The dog studies using systemic GLP-1 infusions provide direct 
guidance as to the expected outcomes. Along with the pacing-induced cardiomyopathy the dogs acquired 
 
Figure D1. Effects of 
combination native GLP-1 
and insulin (1uM) on 
myocardial glucose uptake 
(MG) in the dog model of 
dilated cardiomyopathy 
(DCM). From (12) 
 
Saline 
GLP-1 
Heart Rate (bpm) 
65.0±8.2 
61.8±8.2 
Mean Arterial Pressure (mmHg) 
80.5±6.6 
83.8±6.5 
Stroke Volume (mL) 
110.8±24.2 
94.3±17.4 
Cardiac Output (L/min) 
6.4±1.3 
5.8±1.2 
Table C1. Hemodynamic effects of GLP-1. N=4, all comparisons 
p=NS. Measurements were acquired noninvasively using 
impedance cardiography and an automated blood pressure cuff. 


 
11
myocardial insulin resistance (Figure D1). The combination infusion of insulin and native GLP-1 produced a 
marked augmentation in myocardial glucose uptake, at least 2-fold greater than that seen with insulin alone 
even prior to the onset of insulin resistance (25) and clearly overcoming this cardiomyopathy-related 
myocardial insulin resistance.  
On the basis of these data and supporting cellular and tissue data as described previously, we predict that the 
combined exposure to liraglutide and insulin will produce greater effects to stimulate myocardial glucose 
uptake than either agent alone. A statistically significant difference between combination treatment and the 
individual treatments will be interpreted as evidence for a significant additive interaction of GLP-1 and insulin 
on myocardial glucose uptake. Conversely, a lack of difference will be interpreted as evidence against an 
important additive effect of the combined exposure. The data generated will also be directly informative 
regarding the individual effects of insulin and liraglutide on myocardial fuel selection, regardless of whether the 
anticipated additive effect is observed. These studies have the advantage of providing scientifically and 
clinically meaningful data regardless of whether the hypothesis is proven or disproven.  
D4. Troubleshooting/Alternative Approaches In generating the data presented above, we did not encounter 
any significant technical problems in the execution of study protocols. The prior success of our team with more 
complicated PET studies of myocardial physiology using insulin infusions and a technically more challenging 
tracer of fatty acid metabolism (described in detail below) argues that we will be able to readily overcome any 
technical barriers that arise.   
It is possible that our estimated effect sizes and/or predicted measurement variability are incorrect. We plan an 
interim assessment of observed effect size and variability after 4 subjects have been studied with each 
exposure, to ensure that our sample size estimates are correct, and to allow modification of study plans if 
necessary.  
There is extensive clinical experience to indicate that the liraglutide will be well tolerated. In order to avoid 
problems with sudden toxicity on the morning of the study on first exposure to liraglutide, we plan to taper 
subjects up to the exposure dose over a brief period in advance of the planned PET study (details below). If 
subjects experience intolerance on the morning of the study despite these efforts, their participation will be 
discontinued in order to avoid potential confounding effects of the nausea response on our measurements.  
Subject recruitment will be by advertizing in local media, and by direct recruitment in the PI's clinic. Over the 
past 12 years we have been consistently successful with recruitment into physiologic studies such as those 
proposed, and in this time we have studied over 350 subjects in various CRC-based protocols. Recognizing 
that recruitment is often the limiting factor for clinical studies of any sort, we have allocated sufficient resources 
in the budget of this proposal to provide adequate personnel time and resources for recruitment and screening 
of subjects. We are extremely confident in our ability to recruit subjects for this study. 
D5. Protocol Details 
We will study only Type 2 diabetic subjects under this protocol. In view of the current widespread use of 
metformin as a first line therapy in diabetes, we will study the effect of liraglutide and/or insulin detemir on a 


 
12
background of metformin treatment. After successfully meeting screening criteria (described below) and 
achieving a stable and well tolerated dose of 2000 mg per day of metformin, subjects will be randomly 
assigned to added treatment with insulin detemir alone, liraglutide alone, or combined insulin detemir and 
liraglutide (using a preset randomization schedule ensuring balanced assignment to all 3 groups). Subjects 
who fail to complete the study will be replaced with the next enrolled subject. 
Subject Selection: Subjects will initially be evaluated over the telephone according to the inclusion and 
exclusion criteria outlined in Table D1. Those who appear to qualify will be invited for a formal screening visit. 
Following a full discussion of the protocol and the nature of the planned studies, subjects will be asked to sign 
the informed consent document. They will 
then undergo screening medical history and 
physical examination, including EKG and 
blood work to confirm eligibility 
Subject Preparation: Subjects will be 
instructed on following an American Diabetes 
Association-compliant diet (1800 kcal, 30% 
carbohydrate, 40% fat, 30% protein). 
Subjects will discontinue their current 
antidiabetic medications and be switched to 
metformin on an increasing regimen targeting 
2000 mg/day during an initial 2-week run-in 
period (subjects will be excluded if unable to 
tolerate at least 1500 mg/day). Metformin 
treatment will continue throughout the 
remainder of the study period. Subjects will 
monitor their blood glucose levels 4 times 
daily, and report these readings to the study 
team at regular intervals for safety and efficacy surveillance. Other medications (e.g. ACE inhibitors, 
angiotensin receptor blockers, HMG Co-A reductase inhibitors) will be withheld the morning of the PET 
measurements, but will continue otherwise. Subjects will be asked to withhold ASA for 3 days prior to the 
planned studies. Participants will return to their prior medication regimens immediately following completion of 
the PET studies (if indicated, the PI may make specific clinical recommendations for improving their treatment 
at that time).   
Subjects assigned to liraglutide treatment will undergo a standard dose-escalation regimen as recommended 
in the product insert, targeting the maximal approved dose of 1.8 mg per day. Subjects unable to tolerate 1.8 
mg/day will be excluded from the study and replaced. Liraglutide will be administered each morning, to align 
with the planned administration on the morning of the PET study.  
Inclusion Criteria: 
Age 18-50 yrs 
BMI >25 kg/m2. HbA1c 7.0-10.0%. Treated with up to 2 oral agents 
Exclusion Criteria:  
Chronic illnesses or infections (other than diabetes mellitus). 
Known coronary artery disease, structural heart disease or abnormal 
ECG on screening evaluation. 
Treatment with more than 2 antihypertensive agents or blood pressure 
>140/95 on two occasions during screening evaluations  
History of claustrophobia, musculoskeletal or other factors which would 
result in an inability to comfortably remain within PET scanner gantry 
for the duration of the imaging protocol  
Occupational, investigational or other known radiation exposure which, 
together with the planned radiologic studies, will result in greater than 
500 mrem total exposure in a 12 month period   
For female participants, current pregnancy 
Treatment with a GLP-1 agonist or DPP4 inhibitor within the past 6 
months 
Known intolerance to injected GLP-1 agonist 
Personal history of pancreatitis, personal or family history of medullary 
thyroid carcinoma, or other contraindications to Liraglutide treatment 
Treatment with a thiazolidinedione class antidiabetic agent within the 
past 6 months 
Recognized microvascular complications (neuropathy, nephropathy, 
retinopathy) 
Table D1. Inclusion/Exclusion Criteria 


 
13
Subjects assigned to insulin detemir treatment will begin treatment with an initial dose of 10 units divided as a 
twice daily injection, and then in concert with the PI will have the dose adjusted to achieve consistent morning 
(fasting) blood glucose readings below 130 mg/dL (in order to align with the anticipated mean level achieved 
with the fixed dosing of liraglutide administered). If participants experience blood glucose readings below 70 
mg/dL more than twice per week, or any reading below 50 mg/dL, the detemir dosing will be reduced even if 
this means they no longer achieve the targeted fasting readings. Insulin detemir will be administered once daily 
in the morning, including the morning of the PET study. 
PET protocol: The effects of each hormone exposure on myocardial fuel selection will be studied using 
positron emission tomography (PET). We will measure cardiac perfusion and oxidative metabolism (11C-
acetate), fatty acid uptake and oxidation (11C-palmitate) and glucose uptake (18F-deoxyglucose) under resting 
fasting conditions with hormone exposure (See Figure D1). Blood samples for later measurement of circulating 
levels of relevant metabolites (glucose, insulin, glucagon, free fatty acid, lactate, liraglutide) will be taken at 
planned intervals across the study. Noninvasive measurements of systemic hemodynamics will also be taken 
simultaneously during the PET studies, at similar intervals using surface electrodes placed on the thorax and 
neck, and an automated blood pressure cuff (impedance cardiograph, BioZ.com, CardioDynamics, San Diego).  
Study Day Procedures: Subjects will be admitted 
to the Indiana University Clinical Translational 
Sciences Institute's Clinical Research Center 
before 0700h the morning of Day 1, following an 
overnight fast (from 2000h the day prior). A 20G 
X 3cm Teflon coated catheter will be placed in 
the antecubital vein of one arm, for GLP-1 and 
other infusates. A second 20G X 3cm Teflon 
coated catheter will be placed in the contralateral 
arm for blood sampling.  
Subjects will administer their ongoing doses of liraglutide and/or insulin detemir by 0800h. At ~0900h, subjects 
will be asked to void their bladders prior to being positioned in the scanner and undergoing instrument 
calibration scans. Active scanning will begin ~1000h with the radiotracer injection and scanning protocol as 
outlined in Figure D2.  Immediately after the scanning protocol is completed, subjects will void their bladders 
again, so as to minimize bladder radiation exposure related to concentration of tracers in the urine. After end-
of-protocol blood samples are obtained, subjects will be fed lunch and discharged from the Clinical Research 
Center. At that time the PI will provide recommendations to each participant regarding their ongoing diabetes 
management and arrange follow-up if necessary. 
D6. Statistical Methods  
Study Endpoints The primary study endpoint will be myocardial glucose uptake, measured using PET. 
Prespecified secondary endpoints of interest will include the other PET kinetic parameters of myocardial fatty 
 
Figure D2. Study schema for the PET imaging protocol. Hormone 
exposures begin 2 to 4 hours prior to PET measurements, varying 
by agent, and continue throughout the imaging protocol. PET 
measures take place with infusion of each tracer, followed by a 
washout period to allow counts to return to background levels. 
Arrows indicate time points where hemodynamic measurements 
and blood sampling will take place. Transmission scanning in 
advance of the tracer infusion is not depicted.  


 
14
acid uptake and oxidation, total myocardial oxidation, and myocardial blood flow. Other endpoints of interest 
include rate-pressure product, systemic hemodynamics and changes in circulating levels of metabolic 
substrates and hormones. The primary endpoint and other fuel kinetics endpoints will be adjusted for changes 
in demand if significant changes in cardiac work or total oxidation are observed. 
PET Endpoints for Analysis Myocardial glucose uptake will be quantified from the time-activity curve 
following 18FDG injection using a 3-compartment model, according to the methods of Morita and colleagues 
(76), with a lumped constant of 1.0 (77-79), as was done for the preliminary data above. Tissue perfusion and 
total oxidative metabolism will be quantified from the myocardial time-activity curve following 11C-Acetate 
injection, using a well-validated two-compartment model (80, 81). The influx parameter k1 will be used as the 
measure of tissue perfusion, and the clearance parameter k2 will be used as the index of total oxidative 
metabolism. Fractional tracer retention will be used to calculate fatty acid uptake, and a 3-compartment model 
for fatty acid kinetics will be used to derive rates of fatty acid uptake and oxidation from the time-activity curves 
(82-84).  Normalizing transformations of these outcome variables will be applied if appropriate, depending on 
the observed distribution of data collected. 
Statistical Approach We are specifically interested in whether the combined exposure produces values 
greater than exposure to liraglutide alone; therefore the main comparison will be an unpaired t-test across 
these two treatment groups. One-way ANOVA or mixed modeling will be used to undertake this comparison 
with adjustment for covariates of interest, recognizing that only a limited set of such analyses is possible with 
the comparatively small sample size. Parallel analyses will be undertaken to evaluate effects of liraglutide 
alone versus combination exposure. We also plan to undertake exploratory analyses using multiple linear 
regression to evaluate relationships of the primary and secondary endpoints with the other measured variables 
(e.g. between myocardial 
glucose uptake response 
and circulating hormone 
and metabolite 
concentrations). We 
have not made 
adjustments to sample 
size estimates for these 
exploratory analyses. 
Sample Size 
Considerations 
Published rates of basal myocardial glucose uptake measured using 18FDG in control populations under fasting 
conditions range from 10-60±5-20 μmol/100g/min and insulin-stimulated rates are 100-120 ±30 μmol/100g/min 
(85, 86) (i.e. overall SD ~1/3 to 1/2 of basal value and ~1/4 of the delta with insulin). In our preliminary studies 
we observed mean values at the low end of this range, but proportionally comparable variability (Table 
D2).  The dog studies reviewed above established that GLP-1 (1.5 pmol/kg/min) can produce a doubling of 
 
Baseline 
rates 
(mean±SD) 
Stimulated 
rates 
(mean±SD) 
Demonstrable 
Effect Size 
n=9, 
power=0.8 
Demonstrable 
Effect Size 
n=9, power=0.9 
MGU (µmol/100g/min) a 
11.9±8.7 
25.7±15.6a 
21.9 
25.4 
MBF (mL/100g/min) a 
67.0±5.3 
85.0±32.1a 
45.2 
52.3 
MVO2 (mL/min) a 
26.9±5.4 
37.4±6.4a 
53.3 
60.9 
MFAO (nmol/g/min) b 
34.4±20.2 
4.8±1.2b 
1.7 
2.0 
Table D2. Effect sizes and variability observed in our preliminary studies for the primary 
and main secondary endpoints. The effect size is estimated for the main comparison of 
interest, namely the comparison between combination treatment and insulin alone. Effect 
sizes estimates are calculated for a 2-sided alpha= 0.05. MBF, myocardial blood flow; 
MFAO, myocardial fatty acid oxidation; MGU, myocardial glucose uptake; MVO2, 
myocardial oxygen consumption. Superscript a indicates these values are calculated 
based on observed effects of GLP-1. Superscript b indicates these values were calculated 


 
15
myocardial glucose uptake (12, 29), and our preliminary data are concordant with this effect size (Figure D4 
and Table D3).  
For the other metabolic parameters of interest, overall relative effect sizes and measurement variability are of 
comparable scale to those of MGU (Table D2 and Figure C4). Therefore sample sizes derived from 
myocardial glucose uptake measures are likely adequate for simultaneously demonstrating meaningful 
changes in these secondary endpoints. 
For our outcome of interest we need power to demonstrate a between-treatment difference on the order of the 
effect seen with insulin alone (i.e. at least a doubling of the effect). This is approximately what has been seen 
in dog studies evaluating interactions of GLP-1 and glucose (25).  Using MGU as our endpoint for these 
calculations, and using a two-group t-test design with pooled variability estimates, we calculate that 9 subjects 
per group will provide at least 80% power to demonstrate a between-condition difference of 21.9 
µmol/100g/min in myocardial glucose uptake with a 2-tailed p value <0.05. This represents a demonstrable 
increase in MGU of at least ~80% over the effect of insulin alone. With this sample size we predict we will have 
90% power to demonstrate the anticipated doubling of effect with combination treatment compared to insulin 
alone. Similar calculations provide projections of demonstrable effect sizes for the main secondary endpoints 
(Table D2). (Power estimates used online calculators at http://www.stat.uiowa.edu/~rlenth/Power/index.html.) 
Due to the high cost of the study measurement procedures, we plan to replace subjects who drop out of the 
study (again applying the randomization scheme) rather than initially study a larger number in anticipation of 
drop-outs.  
Data are recorded and stored in a custom-designed SQL database system hosted on our institution's server 
farm. This system meets HIPAA and FDA mandated standards for data security. 
D7. Technical Details 
PET Imaging A Siemens ECAT EXACT HR+ whole body PET scanner is available in the Department of 
Radiology for research studies. This system has an intrinsic in-plane image resolution of approximately 4.2 mm 
FWHM (full width at half maximal). The system simultaneously measures 63 image planes with an axial 
coverage of 15.5 cm. A Siemens CTI RDS 112 medical cyclotron system is used for the production of 
radionuclides.  The Indiana University radiopharmacy is a member of PETNET, a radiopharmacy network that 
produces 18F- deoxyglucose commercially for clinical use, with the attendant high-throughput, expertise, and 
rigorous quality controls this entails (www.petnetsolutions.com).  11C and 18F will also be generated using this 
cyclotron, and the compounds will be labeled as previously described (80, 87, 88). The labeled chemicals are 
collected, evaporated to dryness, reformulated in isotonic NaCl solution and filtered through a 0.22μm filter 
(Millex-GS, Millport, Bedford MA). Radiochemical purity by HPLC, pyrogenicity and sterility tests are performed 
on each product batch.  Conventional data acquisition procedures will be used to acquire and reconstruct the 
PET images, as previously published (89). The image data will be reconstructed using conventional filtered 
backprojection algorithms and a Hanning smoothing filter, which produces an image resolution of 
approximately 1.0 cm FWHM. Computer-based parameter estimation compartmental modeling algorithms are 


 
16
applied to the serial tracer intensity data, to extract parameters which reflect the kinetics of appearance or 
disappearance of the radionuclide.   
Impedance Cardiography: Systemic hemodynamic parameters will be measured noninvasively using 
impedance cardiography (BioZ, CardioDynamics, San Diego CA) (89, 90). The change in impedance across 
the thorax over time (dZ/dt) reflects the cardiac cycle, and the morphology of the waveform allows calculation 
of stroke volume (time-frequency distribution), peak blood flow rates (dZ/dt max) and contractility (upslope of 
dZ/dt) (90, 91).   
Hormones: Insulin (Humulin R, Lilly, Indianapolis IN) will be diluted in 100 mL NS and delivered at 40 
mU/kg/min. Liraglutide will be provided by Novo Nordisk A/S (Copenhagen, Denmark) in clinically available 
pens allowing injection of 0.6 and 1.8 mg doses. 
Substrate and Hormone Measurements: Plasma glucose will be measured by the glucose oxidase method 
(STAT3000 Yellow Springs Instruments, Yellow Springs OH). Non-esterified fatty acid concentrations will be 
determined by a colorimetric assay using a kit from Wako (Richmond, VA). Glucagon will be measured using 
an EIA kit from Millipore (Billerica, MD). Samples for liraglutide and GLP-1 measurements will be collected into 
blood tubes pre-treated with DPP-4 inhibitor (Linco DPP4-010). GLP-1[7-36]amide will be measured using 
ELISA (Millipore), detection limit 3 pM and no cross-reactivity  to GLP1[9-36], GLP-2 or glucagon. GLP-1[9-36] 
will be measured using a non-specific GLP- 1 C-terminus assay (Millipore;detecting both 7-36 and 9-36 forms), 
followed by subtraction of the measured amount of active GLP-1 . Insulin will be measured by ELISA (Alpco, 
Salem, NH).   
D8. Timeline 
Studies will be performed in random sequence according to the randomization scheme generated at study 
inception. We anticipate completing these studies within 12 months. 
D9. Investigational uses of study drugs 
The proposed studies will apply Insulin Detemir and Liraglutide in a manner outside of the current FDA 
approval. Specifically, liraglutide is not FDA indicated for use in combination with any form of insulin. Therefore 
the PI will perform this work under Investigational New Drug surveillance by the FDA, to be obtained by the PI 
for this project. 
 
 


 
17
 
E.  Human Subjects 
E1. Risks to the Subjects 
a. Involvement and Characteristics of Subjects 
The characteristics of the study populations are described in the section on experimental design and methods. 
Lean healthy subjects of both genders will comprise the study group for the work proposed. All subjects will be 
between the ages of 18 and 50 years and healthy. No children under age 18, fetuses, pregnant women, 
prisoners or mentally disabled individuals will be studied. Study populations will include both genders and all 
racial groups, although the study design is not directed at detecting gender or racial differences. All data 
obtained will be for research purposes only.  
Study population to be recruited: 
Volunteer population of Marion County, IN 
 
 
American 
Indian or 
Alaskan 
Native 
n (%) 
 
Asian or 
Pacific 
Islander 
n (%) 
Black, 
not of 
Hispanic 
Origin 
n (%) 
 
Hispanic 
 
 
n (%) 
White, not 
of 
Hispanic 
Origin 
n (%) 
 
Other or 
Unknown 
 
n (%) 
 
Total 
 
 
n (%) 
Female 
 
 
3 (22.1) 
0 (1.05) 
10 (78.5) 
 
13 (52.2) 
Male 
 
 
4 (20.1) 
0 (0.95) 
10 (76.3) 
 
14 (42.8) 
Total 
 
 
7 (21.2) 
0 (1.0) 
20 (77.3) 
 
27 (100) 
Source: Marion County: US Bureau of Census 
 
b. Sources of research material 
Lab specimens: Blood and urine will be collected for routine screening chemistries and hematology to rule out 
disease at the time of screening. At timed intervals during the studies blood will be collected for glucose, 
insulin, non-esterified fatty acid and GLP-1 concentrations. Extra blood will be stored for possible later 
biochemical analyses (no genetic material will be extracted or stored).  
Hemodynamic measurements: cardiac output, heart rate, blood pressure, systemic vascular resistance, and 
other parameters derived from combinations of these measures by impedance cardiography 
PET measurements: basal and hormone-stimulated myocardial glucose uptake, total oxidation rates, and rates 
of myocardial blood flow. 


 
18
Metabolic Measurements: oral glucose tolerance, percent body fat and lean muscle mass. 
c. Potential Risks  
The potential risks to the subjects associated with insertion of catheters include a slight (~1/1000) risk of 
bleeding/hematoma and infection. Regarding avoidance of infection, meticulous care will be taken with respect 
to sterility and technique. The catheters will be kept patent with saline to prevent clotting.  
PET scan – Subjects with a history of claustrophobia will be excluded from participation due to the somewhat 
close nature of the gantry of the PET scanner. There is a routine period of ~30 min for transmission scans prior 
to the first radionuclide injection; subjects’ comfort will be routinely assessed at that point prior to continuing. 
The use of radionuclides results in radiation dosing to the research participant.  20 mCi of 11C-acetate will be 
used. This produces an estimated total body dose of 40 mrem, with the maximal individual organ dose of 1480 
mrem in the pancreas, compared to the annual occupational limit of 50,000 mrem for the pancreas. 5 mCi of 
18FDG will produce a whole body dose of approximately 340 mrem, with a maximal organ dose of 160 mrem in 
the bladder, compared to the annual occupational limit of 50,000 mrem for the bladder. 7.5 mCi of 11C-
Palmitate will produce a whole body dose of approximately 44 mrem, with a maximal organ dose of 3,850 
mrem in the bladder. In total for the complete study subjects will receive approximately 424 mrem. By 
comparison, a typical radiation dosage from an AP chest film is 30 mrem, and the maximal permissible whole-
body dose for the general population in research applications is 500 mrem/year. Radiation exposure confers 
significant risks to developing fetuses, and therefore all female study subjects with the potential to be pregnant 
will be specifically tested immediately prior to PET protocols and excluded if pregnant.  
Risks from the treatments: Liraglutide is an FDA-approved treatment for diabetes with a well-understood side 
effect profile. A minority of subjects will be expected to experience some GI discomfort on initiation of therapy, 
which is expected to abate. The standard dose escalation procedure is designed to help patients tolerate this 
effect and allow time for them to adjust to receiving the medication. Some subjects will be unable to tolerate 
the full treatment dose, and will be withdrawn from the study if that is the case. Other common side effects 
affecting >5% of treated subjects include diarrhea, headache and vomiting. As with the nausea these are 
expected to abate. In rat models GLP-1 based treatments are associated with medullary carcinoma of the 
thyroid, a rare form of thyroid cancer. There is insufficient data to conclusively rule out this effect in humans, 
and therefore subjects with increased risk for this due to a personal or family history of predisposing conditions 
will be excluded. GLP-1 based treatments have been associated with an increased prevalence of recognized 
pancreatitis (although there is debate whether this represents a drug treatment effect or a previously 
unrecognized problem among patients with diabetes). Participants with a known history of pancreatitis will be 
excluded from participation. Hypoglycemia is a possibility in participants where liraglutide is added to other 
glucose-lowering agents; the rate of hypoglycemia in subjects treated with liraglutide alone is very low. All 
subjects will be routinely monitoring their blood glucose and instructed on the appropriate management of 
hypoglycemia.  


 
19
The risks of insulin treatment are well recognized, and no different when using insulin detemir than other 
insulin formulations. The most common and important problem is hypoglycemia. As above, this will be 
managed by having subjects monitor their blood glucose levels routinely.  
As with all human subject-based medical research, there is a risk of loss of confidentiality owing to disclosure 
of health information. These disclosures may be mandated by local or national regulatory agencies (i.e. IRB, 
FDA) or may be unintentional. Safeguards regarding both physical and electronic copies of all subject data are 
in place, as dictated by the current standards of Indiana University.  
E2. Adequacy of Protection Against Risks 
a.  Recruitment and Informed Consent Procedures 
All personnel associated with this study have successfully completed specific training in Human Subjects 
Protection, as required by our local IRB and HHS. All subjects will be recruited directly and by advertisement 
from the general local population and the various clinics at IU Medical Center and the Indianapolis Veterans 
Affairs Medical Center which serve the general population of Marion county and surrounding areas. All 
volunteers will be enrolled irrespective of race, creed and sex, therefore, one would expect that the study 
population mirror the demographics of the area, i.e., approximately 21% Afro-American, less than 2% Hispanic, 
American Indian and Asian, and 77% Caucasian with equal gender distribution. When we look at our 
recruitment statistics over the past few years including all studies  (NIH and other funding), we find that we 
recruit ~40% women, and 49% black, 50% white and 1% Hispanic. Thus, we have successfully met federal 
mandates for representative inclusion of study participants with our recruiting strategy. The PI is cognizant of 
the recurrent problem of obtaining equal populations of both genders and therefore we continue to 
aggressively recruit to meet that goal. 
Prior to enrollment in the study, the entire protocol will be reviewed with each volunteer. Subjects will be given 
ample opportunity to ask questions, and once all concerns are satisfied, formal written consent will be 
obtained. A signed consent form will then be saved as a permanent record. No studies will be initiated until 
approval is obtained by the Indiana University-Purdue University at Indianapolis (IUPUI) Institutional Review 
Board (an OHRP-registered IRB) and Radiation Safety approval. Subjects will be compensated financially for 
their participation.  
b.  Protection Against Risks 
During these studies there are medically trained personnel at the bedside and the subjects are monitored 
continuously. In the unlikely event that any of the above mentioned complications occur, immediate medical 
intervention would be undertaken.  
In menstruating females, pregnancy will be ruled out by urine pregnancy test immediately prior to study. Such 
subjects can only participate with a documented negative test immediately prior to initiation of the study 


 
20
Risks associated with the treatments will be managed overall through controlled dosing exposure, and through 
close monitoring of blood glucose across the course of the treatment period. Subjects at particularly elevated 
risk of the rare complications associated with liraglutide treatment will be excluded at the screening stage.  
The risks associated with radiation exposure are managed through limitations of exposure for non-clinical 
reasons. The limits applied to research subjects are arbitrarily set at 1/10 of those allowed for occupational 
exposure, namely 500 mrem/calendar year. Subjects’ recent prior exposure will be assessed during screening 
for participation in this protocol, and if their prior exposure plus the planned studies will exceed this limit they 
will be excluded from participation. 
Except for research data, all other information relevant to patients collected in these studies will be kept 
confidential, including locked storage of all paperwork, and secure digital storage on a database to which only 
members of our research group have access. The security and integrity of this database are maintained with 
the direct assistance and oversight of the Department of Medicine’s Information Technology support teams, 
and the database is served centrally from a closely monitored, regularly backed up server. The current HIPAA 
and FDA standards for research database security are met by these efforts. 
E3. Potential Benefits of the Proposed Research to the Subjects and Others 
The subjects will not benefit directly from participating in this study except possibly for a physical examination 
and determination of lean and fat body mass performed prior to the study. In general, the benefit of the PET 
quantification of heart fuel selection relates to the favorable signal-to-noise ratio, which allows us to undertake 
studies of human physiology in very small numbers of subjects. Therefore, the benefit to society related to 
each individual’s participation is maximized.  The potential benefit to society in general is a better 
understanding of the role and application of GLP-1 in the modulation of myocardial fuel selection, with the 
intent to further translate these observations into clinical practice. This has a clear significance to the 
population as a whole, given the dominant role of cardiovascular disease in morbidity and mortality in North 
America and the world. 
E4. Data Safety Monitoring Board 
Composition of the Data and Safety Monitoring Board  
The DSMB for this protocol will consist of the PI and the co-investigators, and will be chaired by Dr. A Lteif 
(Endocrinology/Medicine)(Chair of the DSMB) who is not directly involved in the study. 
Frequency of Review 
The Principal Investigator will make a report to the DSMB at 6 month intervals, and the DSMB will meet with 
the PI to review this report. 
Features Assessed 
Adverse Events Grading and Attribution Adverse events in this protocol are expected to be evident during the 
hospitalization for the study measurements. Study subjects are encouraged to contact study staff with any 


 
21
concerns following discharge from hospital, and are assessed on the GCRC at the discretion of the study 
physician.  
The PI, in consultation with the DSMB, will grade adverse events severity as follows (as recommended by the 
National Cancer Institute): 
0-No adverse event or within normal limits 
1-Mild adverse event 
2-Moderate adverse event 
3-Severe adverse event 
4-Life-threatening adverse event 
5-Fatal adverse event 
Grade 4 and 5 events will prompt an immediate report to the DSMB, with expedited reporting to the IRB and 
the GCRC (see below). 
Where investigational agents or devices are used, attribution of the adverse event to this agent or device will 
be categorized as follows (as recommended by the National Cancer Institute): 
Definite – The AE is clearly related to the agent or device 
Probable – The AE is likely related to the agent or device 
Possible – The AE may be related to the agent or device 
Unlikely – The AE is doubtfully related to the agent or device 
Unrelated – The AE is clearly NOT related to the agent or device 
Protocol Compliance and Data integrity: Any deviations from protocol will be reported to the DSMB. 
Completeness and accuracy of data records will be assessed by a random check of 5 recent study records. 
Data sufficiency/Study completion: Assessments of completeness of data collection for subsections of this 
project are continuous, and will be reported by the PI at each meeting.  
Protocol review: Where appropriate, changes to the protocol suggested either by interim results, adverse 
events, or external events such as new FDA recommendations will be discussed with the DSMB. 
Reporting 
Any serious and unexpected adverse events associated with the study intervention that occur on-site will be 
reported to the IRB and the GCRC within 3 working days of notification of the event. Any serious and 
unexpected adverse events associated with the study intervention that occur at an external site will be reported 
to the IRB and the GCRC within 10 working days after receipt from the sponsor (this statement is a generic 
IUPUI IRB requirements, does not apply to this study). All other adverse events will be reported annually with 
the continuing review. All expedited reports will also be sent to the GCRC. 


 
22
Biohazards 
Potential hazards related to infusates (GLP-1, insulin) and to radiation exposure (PET scanning, DEXA 
measurement of body composition) are addressed in detail above under E3c (Potential Risks) and E4b 
(Protection Against Risks). This project will be performed under FDA Investigational New Drug supervision 
(IND 105,155).  
E5. Women and Minority Inclusion in Clinical Research 
Women and minorities will be studied under this proposal, in proportions anticipated to reflect the proportions 
in our local population (see recruitment population table above). 
E6. Inclusion of Children 
Recruitment criteria include subjects aged 18 and above who otherwise meet the study criteria. Cardiac fuel 
selection across the stages of puberty has not been studied, and in particular it is unclear whether sex steroids 
are potential regulators. Therefore, studying children who are traversing puberty could introduce confounders, 
and specifically asking this question requires an entirely different study design. Therefore, we plan to include 
post-pubertal children only, aged 18 and above. 
 
F. Vertebrate Animals 
N/A 
 
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