Clinical trial NCT01951339
Conditions: Type 2 Diabetes Cardiovascular Disease
Keywords: diabetes type 2 diabetes exercise echo heart sitagliptin glimepiride cardiovascular
Sponsor: University of Colorado, Denver
Start date: 2013-10-01; Completion date: 2018-06-01
Enrollment: 36 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The goal of this study is to examine whether sitagliptin, an agent which enhances the action of hormones that control the release of insulin and is already in clinical use for type 2 diabetes, might also improve functional exercise capacity.

Specific aims:

1\. To test whether sitagliptin will improve functional exercise capacity in persons with type 2 diabetes compared to glimepiride.

1a. The primary outcome will be peak oxygen consumption (VO2peak) and oxygen uptake kinetics (VO2 kinetics).

1b. Secondary outcomes include cardiac function, endothelial function and tissue oxygen saturation (STO2) as well as health-related quality of life.

2\. To evaluate the impact of sitagliptin on muscle mitochondrial function 2a. The primary outcome to address this aim will be 31P measurements (phosphocreatine, free inorganic phosphate, adenosine triphosphate peaks, adenosine diphosphate and pH)

Impact: Novel approaches are needed to decrease excess cardiovascular morbidity and mortality in diabetes. Diabetes impairs cardiovascular fitness and thereby mortality. A demonstration that sitagliptin improves cardiovascular fitness, (and possibly mitochondrial function) will provide important new data pertinent to the management of diabetes and pre-diabetes.

Detailed description:
Subjects will come for a total of nine testing visits during which evaluations will take place. Visits are structured as follows:

1. After subjects review the study and give consent for study participation, a history and physical exam will be performed. Ankle brachial index, autonomic nervous system function tests, the Low-level Physical Activity Recall questionnaire and vital signs will be performed.
2. Blood drawn for measurement of hemoglobin A1C, fasting glucose, fasting insulin, free fatty acids and microalbuminuria, c-reactive protein, interleukin 6, adiponectin, and creatinine and glycerol. Additional screening labs include complete blood count (CBC), follicle-stimulating hormone, urine protein and a lipid panel to assess whether women are pre- or post-menopausal (FSH), and overall health (CBC, lipids and urine protein). A dietary survey will be administered for food preferences for the three day study diet administered prior to visits 3-5 and 7-9. Dual-energy xray absorptiometry (DEXA) and body composition tests will be done to ensure that groups are weight similar (using fat-free mass). A pulmonary function test, resting electrocardiogram (EKG) and familiarization bicycle test will be performed.
3. Subjects will receive a three day study diet prior to visit 3. A resting and exercise EKG will be performed on the day of the visit. A graded exercise test will be done to determine the VO2peak. Patients will have measures of cardiac function and endothelial function on visit 3 by plethysmography and cardiac echo. Vital signs will be taken at rest.
4. Subjects will receive a three day study diet prior to visit 4. Calf muscle magnetic resonance spectroscopy (MRS) will be performed on a 3.0 T whole-body MRI scanner.
5. During visit 5, arterial stiffness/endothelial function will be non-invasively measured by the Sphygmocor system. Subjects will also have three constant-load tests to measure VO2 kinetics where oxygen saturation (StO2) will be measured during exercise. A resting and exercise EKG and vital signs will be performed during the visit. Subjects will be randomized to taking sitagliptin plus placebo or glimepiride plus placebo and all must be taking metformin (1-2 grams /d) for 3 months. Sitagliptin and its placebo will be administered 100 mg/d. Glimepiride and its placebo will be administered 2 mg/day. During the treatment phase subjects will be given a log to keep track of their blood glucose each day.
6. Visit 6 will consist of a physical exam with a clinician as well as a blood draw and check of vital signs during sitagliptin or glimepiride treatment.
7. After 3 months of sitagliptin or glimepiride administration, Visit 3 will be repeated. Additional testing to be performed during visit 7 will include a physical exam performed by a study physician, blood work for covariate lab tests listed in Visit 2 and the Low-level Physical Activity Recall(LoPAR) questionnaire.
8. During visit 8, visit 4 procedures will be repeated.
9. During visit 9, the testing performed during visit 5 will be repeated.

Arms: arm 1: 100 mg sitagliptin plus 2 mg placebo once daily for three months arm 2: 2 mg glimepiride plus 100 mg placebo once daily for three months
Interventions: intervention 1: Sitagliptin intervention 2: Glimepiride intervention 3: Placebo intervention 4: Placebo
Intervention descriptions: intervention 1: 100 mg sitagliptin intervention 2: Active Comparator

2mg glimepiride intervention 3: 2 mg placebo once daily intervention 4: 100 mg placebo once daily for three months

Protocol documents:
Prot_SAP_000.pdf: 
Protocol Template 
Page 1 
CF-146, Effective 7/10/11 
 
w 
COMIRB Protocol 
 
PROTOCOL #: 
 
13-2015 
 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Project Title: 
Impact of sitagliptin on cardiovascular exercise performance in type 2 
diabetes 
 
 
 
Principal Investigator:  
Judith G. Regensteiner, PhD and Jane Reusch, MD 
Version Date: 09/15/2016 
 
 
 
 
  
 
I. Hypotheses and Specific Aims: Diabetes is a leading cause of cardiovascular disease (CVD) 
and mortality (5,6). One factor potentially associated with the excess CVD in diabetes is the well-
established impairment in functional exercise capacity in people with type 2 diabetes mellitus (T2D) 
compared to healthy controls (10,42,72,75,78,80). The causes of the exercise abnormalities in T2D 
are unknown, although recent work from our group demonstrates associations between insulin 
resistance, endothelial dysfunction, mitochondrial dysfunction and cardiac dysfunction with 
decreased exercise capacity in T2D in humans (4,10,42,59,72,75,77,78,80,94). We have also shown 
in animal work that the normal response to exercise of both vascular mitochondrial function and 
biogenesis regulators is blunted in CVD and diabetic models (43).  
     The long-term goal of our laboratory is to understand the mechanisms whereby T2D impairs 
functional exercise capacity and to develop approaches to improve function.  Observations from our 
group have identified two interventions that improve, but do not normalize, exercise function in T2D.  
These include: supervised exercise training (10) (and see preliminary data) and more recently, 
treatment with an insulin sensitizer (74). This latter study illustrated that drug therapy could improve 
exercise capacity in uncomplicated T2D (74).  Despite improvements, neither of these interventions 
succeeded in normalizing exercise function to equal that of nondiabetic controls. The goal of this 
proposal is to examine whether sitagliptin, an agent which enhances incretin action, and is already 
in clinical use for T2D might also improve functional exercise capacity.   
      Sitagliptin works by inhibiting dipeptidyl peptidase 4 (DPP-4) inactivation of the incretin hormones 
glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This 
increases active incretin and insulin levels, and decreases glucagon levels and postglucose-load 
glucose excursion. Increasing evidence suggests that sitagliptin and other DPP-4 inhibitors may also 
provide protection against CV events (39,54,56), although the mechanism for this is not clearly 
understood. In addition, in a recent study from our lab, saxagliptin restored vascular eNOS and 
mitochondrial induction after an 8 day exercise intervention in a rodent model of diabetes (41). 
      Incretins have additional relevant effects that are of particular interest for this proposal including 
benefiting endothelial and mitochondrial function (22,26), as well as reducing CV risk (51). As has 
been noted, GLP-1 is a critical factor for healthy metabolism and vasculature and stimulates eNOS 
(26), (22).  Small studies in humans with congestive heart failure show improvements in cardiac 
function and exercise capacity with GLP-1 administration (82,83). Improvements in endothelial 
function have also been examined in rodents in vivo and ex vivo suggesting a direct effect of GLP-1 
on endothelial function via eNOS (66-68). In humans, this is manifested in consistently lowered blood 
pressures in some studies (49,50). Thus overall, increasing evidence in humans and animals 
suggests that sitagliptin and other incretin regulatory agents may have some cardioprotective effects. 
Recent observations in rodents and humans suggest that GLP-1 has a beneficial effect on 
endothelial and cardiac function (two factors associated with exercise function in children and adults 
with diabetes) (3,48,49,60,63,82,84,87,88). It is plausible therefore, that sitagliptin will improve 
exercise capacity in this group.  
 
Our primary hypothesis is that sitagliptin will improve functional exercise capacity in people with 
T2D more than glimepiride. Our secondary hypothesis is that sitagliptin will improve 
mitochondrial function more than glimepiride.  


Protocol Template 
Page 2 
CF-146, Effective 7/10/11 
 
 
Specific aims: 
1. To test whether sitagliptin will improve functional exercise capacity in persons with T2D 
compared to glimepiride.  
1a. The primary outcome will be peak oxygen consumption (VO2peak) and oxygen uptake kinetics 
(VO2 kinetics).  
1b. Secondary outcomes include cardiac function, endothelial function and tissue oxygen saturation 
(STO2) as well as health-related quality of life.   
Rationale: In this study, we want to compare two approved active drugs used to treat diabetes. 
GLP-1 improves exercise capacity in persons with heart failure (82,83).  In addition, GLP-1 has 
effects on cardiac and endothelial function in rodents and humans as well as its metabolic effects 
(3,48,49,60,63,82,84,87,88). Saxagliptin restores vascular eNOS and mitochondrial induction after 
an 8 day exercise intervention in a rodent model of diabetes (41). Glimepiride is chosen as an active 
comparator because it is a commonly used oral agent but has no known effect on exercise capacity. 
Approach: We will compare exercise capacity in patients with T2D taking sitagliptin plus placebo vs 
glimepiride plus placebo to determine whether a three month intervention with sitagliptin improves 
VO2peak, oxygen uptake kinetics, cardiac function, blood flow, endothelial function and STO2. These 
experiments will address the CV and functional impact of sitagliptin on exercise in diabetes.  
 
2. To evaluate the impact of sitagliptin on muscle mitochondrial function 
2a. The primary outcome to address this aim will be 31P measurements (phosphocreatine, free Pi, 
ATP peaks, ADP and pH) 
Rationale: It is believed that sitagliptin works by inhibiting the DDP-4 inactivation of incretin 
hormones (GLP-1, GIP). In general, the GLP-1 receptor acts to stimulate eNOS and ceramide 
transfer protein (CERT) thereby activating mitochondrial biogenesis and augmenting glucose flux 
and enhancing oxidative phosphorylation. New data from our group indicates that muscle 
mitochondrial function as assessed by 31P MRS correlates with functional exercise capacity (41). It 
is unknown whether this is related to decreased blood flow and substrate delivery or a specific defect 
in tissue mitochondrial function. Preliminary work suggests that improving oxygen delivery may 
improve mitochondrial function. GLP-1 has been demonstrated to increase muscle blood flow (25).  
It is therefore plausible that eNOS stimulation by sitagliptin will improve muscle blood flow, 
mitochondrial biogenesis and thereby muscle mitochondrial function.  
Approach:  MRS techniques will be used to evaluate mitochondrial function. These experiments will 
generate new information on the impact of sitagliptin on mitochondrial function in human skeletal 
muscle.  
Impact: Novel approaches are needed to decrease excess CV morbidity and mortality in diabetes. 
Diabetes impairs CV fitness and thereby mortality. A demonstration that sitagliptin improves CV 
fitness, (and possibly mitochondrial function) will provide important new data pertinent to the 
management of diabetes and pre-diabetes. 
 
II. Background and Significance:  
Exercise function is abnormal in people with T2D. Even in the absence of cardiovascular disease 
(CVD), persons with T2D have an impaired ability to carry out exercise both at maximal and 
submaximal workloads (10,42,72,75,78,80).  The functional significance of decreased exercise 
performance is that in T2D, poorer exercise capacity is clearly associated with an array of adverse 
health outcomes, ranging from poorly controlled diabetes to increased CV morbidity and mortality 
(5,6). In addition, it has been shown in a population study that there is a correlation between all-
cause mortality and low levels of fitness in T2D as well as in healthy controls (8,9,93).  Therefore, 
improvements in exercise tolerance and thereby fitness in T2D could potentially have multiple long-
reaching benefits for patients.  
     The underlying causes of the exercise abnormalities in T2D are not well known although there is 
evidence available implicating several factors. Importantly, the metabolic abnormalities intrinsic to 
diabetes may play a role in that insulin resistance has been strongly linked to the exercise 


Protocol Template 
Page 3 
CF-146, Effective 7/10/11 
 
impairments observed  (10,12,37,40,44,78).  There are also inverse associations between habitual 
physical activity, VO2peak and insulin resistance (81). Previous studies have reported relationships 
between higher levels of physical activity/exercise training status and improved insulin sensitivity in 
nondiabetic persons as well as in persons with T2D (12,37,40,44,75,78) related to impaired exercise 
capacity in persons with even very recently diagnosed T2D during exercise. Abnormal VO2 kinetic 
and muscle O2 saturation (StO2) findings support the idea that O2 delivery is impaired in T2D (4,75) 
(and see Preliminary data). In addition, impaired endothelial function is likely implicated as a factor 
associated with reduced exercise performance in T2D in that exercise abnormalities could reflect a 
deficient endothelial dilator response to exercise-mediated demand in heart and in peripheral skeletal 
muscle (16,52,76) (see Preliminary Data). Overall, the published experimental evidence suggests that 
metabolic, cardiac and peripheral circulatory abnormalities as well as endothelial dysfunction are 
associated with the impaired functional exercise capacity in T2D.  In the current proposal we will address 
functional differences between the proposed interventions upon the above contributing factors and begin 
to define the relative importance of metabolic abnormalities, endothelial dysfunction, cardiac dysfunction 
and peripheral circulatory abnormalities for causing exercise impairments.  Exercise training has been 
shown to improve to some degree but not to normalize the metabolic, endothelial, cardiac and peripheral 
circulatory abnormalities observed. Thus, the role of sitagliptin could be of key significance to improve 
exercise abnormalities not normalized by exercise training. 
Agents that improve functional exercise capacity: 
We have previously reported that exercise training and rosiglitazone both improve functional exercise 
capacity (10,74). In rodents, the AMP kinase agonist AICAR and resveratrol (57) improve exercise 
endurance (3,88). Animals expressing PPAR delta ligand in the muscle have increased fitness as seen 
with the “marathon mouse” (92). Treatment with a PPAR delta ligand improved exercise capacity in 
combination with exercise training. Thus, there is evidence that pharmacologic intervention is a plausible 
strategy for improving exercise capacity in T2D.   
Evidence that GLP-1 improves metabolic, endothelial and cardiac function as well as exercise 
performance.  In addition to enhancing insulin secretion by acting on pancreatic ß cell receptors, GLP-
1 also binds to receptor sites in other tissues including brain, gut, adipose tissue and the myocardium.  
GLP-1 has been shown to benefit glycemic control in that it lowers glucose level (both fasting and 
postprandial) and HbA1C. (50,55). Studies in rodents and humans show that GLP-1has positive 
effects on endothelial and cardiac function in T2D (17,35,50).  Specific cardiac effects include the in 
vitro finding of increasing the cAMP concentration in cardiac myocytes (91) as well as improved left 
ventricular function in humans, rodents and dogs (48,50,61-64,82,100). In addition, a key cardiac 
abnormality associated with exercise in T2D is diastolic dysfunction (likely related to poor 
metabolism) and this abnormality was not corrected by exercise training although it was improved. 
In a rodent model of obesity and diabetes (db/db-/- mice), sitagliptin decreased elevated myocardial 
fatty acid uptake and oxidation in the diabetic heart suggesting a beneficial myocardial metabolic 
effect of DPP-4 inhibition (45). Of note, GLP-1 null mice develop a cardiomyopathy that strongly 
resembles the fibrotic changes and diastolic dysfunction observed in other rodent models of diabetic 
cardiomyopathy indicating a role for GLP-1 in myocardial health (66). 
      A proof of concept study in humans with congestive heart failure showed improvements in 
exercise capacity with GLP-1 administration (83).   Diabetic and nondiabetic patients with heart 
failure received GLP-1 infusion over a 5 week period.  Primary end points included evaluation of left 
ventricular function at rest assessed by echocardiography and VO2peak measured on the treadmill.  
Left ventricular function improved significantly. VO2peak improved from 10.8±0.9 to 13.9±0.6 
ml/kg/min (P<0.001) in the treated vs the control group.  In addition, the 6 minute walk test improved 
significantly from 232±15 to 286±12 m (P<0.001). Thus, GLP-1 improved functional exercise capacity 
over a relatively short period of time. Although the patients we propose to study do not have heart 
failure, they do have subclinical cardiac abnormalities which correlate with the exercise defects 
observed and may represent early signs of diabetic cardiomyopathy (77). Thus, the concept of 
administering GLP-1 to patients with diabetes provides a promising candidate agent to improve 
exercise function. Improvements in endothelial function have been examined in rodents in vivo and 


Protocol Template 
Page 4 
CF-146, Effective 7/10/11 
 
ex vivo suggesting a direct effect of GLP-1 on endothelial function via eNOS (66). In humans, this is 
manifested in consistent lowering of blood pressure in clinical trials (50). 
GLP-1 improves vascular function and mitochondrial adaptation:  GLP-1 is an incretin protein 
secreted by the gut in response to food intake that augments insulin secretion, decreases glucagon, 
decreases appetite and slows gastric emptying. Analogs of this hormone are currently approved for the 
management of diabetes (90).  Studies in rodents and humans show that GLP-1 has positive effects 
on endothelial and cardiac function in diabetes (1,2,28,71,90).  GLP-1 decreases mitochondrial ROS 
and improves optimal mitochondrial function in myocardium and beta cells (7,13,22). Direct and 
indirect vascular effects of GLP-1 include induction of eNOS, decreased expression of VCAM-1, 
vasodilation, increased cAMP, decreased intimal hyperplasia and blockade of LPS induced vascular 
permeability (1,20,21,26,28,31,38,58).  In diabetic rats, GLP-1 improves endothelial function and 
stimulates CREB (69,70). In addition, GLP-1 improved hepatic steatosis and increased 
cytoprotective autophagy in non-alcoholic fatty liver disease and beta cells (30,97,98). 
 
Saxagliptin restores the vascular response to exercise including eNOS induction and 
mitochondrial biogenesis in diabetes. We recently reported that short term (8 day) exercise 
intervention increases aortic mitochondrial protein expression in control rats, but not in an obese diabetic 
model (in press-appendix). Similarly, in Goto-Kakizaki (GK) rats, we recently showed that the response 
of eNOS, SIRT3, pCREB or mitochondrial protein expression with exercise in diabetes is impaired and 
that intervention with saxagliptin increased these markers as well as mitochondrial response to exercise 
in diabetes, restoring normal mitochondrial adaptation to exercise in a diabetes model (41). This is likely 
the result of the prevention of GLP-1 degradation by saxaglipitin. Therefore, we expect sitagliptin to 
improve exercise function and propose to evaluate the effect of sitagliptin on mitochondrial function in 
humans in the present study. 
 
Significance: 
    Physical activity has a beneficial effect on CV and all-cause mortality. We know that exercise 
training improves functional exercise capacity in persons with diabetes, but does not close that gap and 
bring people to normal levels of fitness. Sitagliptin, with its potential impact on cardiac, endothelial and 
mitochondrial function may restore exercise capacity to non-diabetic levels. Inactivity has adverse effects 
on the diabetic metabolism and increases CV morbidity and mortality; in contrast, increased physical 
activity works beneficially in these regards (8,9,19,93). Thus, from a practical standpoint, a drug 
which improves exercise capacity has the potential to make the performance of exercise more 
feasible for the patient with T2D.  Decreased functional exercise capacity in people with T2D is a 
barrier to physical activity.  If sitagliptin, or any agent employed for glycemia, improves exercise 
capacity, it may beneficially affect the treatment algorithm for persons with T2D. Overall, the findings 
from this proposal may provide important mechanistic insight into therapies which benefit CV, endothelial 
and mitochondrial outcomes for persons with T2D via enhanced exercise capacity. 
 
III. Preliminary Studies/Progress Report: Below we highlight critical published and preliminary 
results that support our hypothesis and demonstrate the feasibility of this project in our laboratory. 
 


Protocol Template 
Page 5 
CF-146, Effective 7/10/11 
 
1.0 Studies from our laboratory that demonstrate that T2D confers exercise dysfunction:   
1.0a Diabetes is associated with decreased VO2peak: It 
has been consistently observed by our lab and others that 
VO2peak is lower in persons with T2D than in nondiabetic 
controls (4, 10, 42, 72, 75, 78, 80) A difference of 
approximately 20% in VO2peak between persons with T2D 
and controls has been documented in these studies. 
1.0b. Diabetes is associated with slowed VO2 kinetics: 
Not only VO2peak, but also submaximal measures of 
exercise are abnormal in T2D including VO2 at submaximal 
workloads (Figure 1) and VO2 kinetics. VO2 kinetics are a 
measure of the rate of adaptation in pulmonary VO2 
following the onset of constant work rate.  
Three phases to the response of VO2 from rest to moderate 
constant-load exercise were proposed (95,96).  At the onset of 
exercise, VO2 from the lungs normally increases abruptly for the 
first 15 sec (Phase I) as pulmonary blood flow increases.  Then 
the VO2 increases exponentially in Phase II with a time constant, 
tau, normally about 30-45 sec representing further increases in 
blood flow and decreased venous O2 content.  Phase II ends as 
gas exchange approaches a steady-state.  Phase III is steady-
state VO2 below the lactate threshold, but not above the lactate threshold (95,96). We observed that 
VO2 kinetics were abnormal in persons with T2D compared to healthy controls (75) as has been 
observed in other groups with abnormal CV responses to exercise (4, 10) . This finding suggests that 
persons with T2D have a reduced rate of circulatory adjustment to an increase in work-load. 
Improving cardiac or systemic CV function with sitagliptin could correct this defect. 
1.0c Studies characterizing the cardiac contribution to exercise dysfunction in T2D: 
Fig 2 addresses likely causes of exercise abnormalities associated with T2D. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2. Cardiac Abnormalities with Exercise in T2D: Is cardiac function impaired during exercise in 
T2D? Ten premenopausal women with uncomplicated T2D (average diagnosed duration of T2D, 
3.6 years) and 10 healthy nondiabetic women of similar age, weight and activity levels were studied 
and data have been reported (77). Participants performed a maximal exercise test while 
instrumented with an indwelling pulmonary artery catheter. On separate days, Tc-99m sestamibi 
myocardial perfusion (SPECT) imaging with rest and exercise was performed in a subgroup of 
diabetic and control patients. VO2peak, measured during catheterization was lower in T2D than 
control subjects (18.7±2.3 vs 22.3±4.2 ml/kg/min, P<0.05). Pulmonary capillary wedge pressure 
(PCWP) (a marker of cardiac stiffness) rose more with exercise persons with T2D than controls 
(146% vs 114% increase, P<0.01). (Figure 3, Table 1).VO2peak was inversely correlated with 
Figure 2. Schema:  
Proposed factors contributing to exercise dysfunction in 
T2D: T2D and prediabetic conditions such as insulin 
resistance are associated with an abnormal metabolic 
environment that induces endothelial, cardiac, and 
peripheral dysfunction. Target organ dysfunction feeds 
back to exacerbate these defects. Ultimately these factors 
result in defects in functional exercise capacity.  
Type 2 DM/IR
Oxidative Dysfunction
Peripheral Muscle 
Dysfunction
Endothelial
Dysfunction
Cardiac 
Dysfunction
Dysmetabolic 
Environment
Acyl FA, Ox. Stress, Glu, FFA
O2 Utilization
O2 Delivery
Slowed VO2 Kinetics
Exercise Capacity
Type 2 DM/IR
Oxidative Dysfunction
Peripheral Muscle 
Dysfunction
Endothelial
Dysfunction
Cardiac 
Dysfunction
Dysmetabolic 
Environment
Acyl FA, Ox. Stress, Glu, FFA
O2 Utilization
O2 Delivery
Slowed VO2 Kinetics
Exercise Capacity
Type 2 DM/IR
Oxidative Dysfunction
Peripheral Muscle 
Dysfunction
Endothelial
Dysfunction
Cardiac 
Dysfunction
Dysmetabolic 
Environment
Acyl FA, Ox. Stress, Glu, FFA
O2 Utilization
O2 Delivery
Slowed VO2 Kinetics
Exercise Capacity
Figure 1. This figure illustrates that VO2 at all 
submaximal work-loads is reduced in 
persons with T2D compared to nondiabetic 
controls of similar age and activity levels 
during graded exercise testing (78).  This is 
an abnormal response to exercise that has 
been observed in other disease states with 
impaired O2 delivery or utilization (4, 
10,60,99). 


Protocol Template 
Page 6 
CF-146, Effective 7/10/11 
 
maximal PCWP (r=-0.62, P<0.05). Blood flow, as assessed by total normalized myocardial 
perfusion index (MPI) from SPECT was lower in persons with T2D than controls. (11.0±3.5xE-9 vs 
17.5±8.1E-9 respectively, p<0.05) and was inversely related to PCWP (R=-0.56).  Since resting 
systolic function by echo was normal, findings are possibly consistent with diastolic dysfunction in T2D. 
However, cardiac output was not reduced in T2D as might have been expected. Thus, there is clear 
evidence of cardiac dysfunction and reduced perfusion in T2D (4) and the impaired cardiac function is 
correlated with impaired exercise capacity. The component of these early cardiac abnormalities in T2D 
related to decreased perfusion could be responsive to sitagliptin. 
 
2.0a Cardiac abnormalities are observed by echocardiography in adults and adolescents 
with T2D. Having first established the presence of cardiac abnormalities using invasive techniques, 
we next validated and are now using noninvasive stress echocardiographic 
techniques to assess cardiac function in a more recent exercise training 
study. We have preliminary data that markers of diastolic function are 
abnormal in T2D such as the E:A ratio, showing that cardiac diastolic 
function was abnormal in people with T2D (E:A 1.33±0.42 controls and 
1.11±0.26 T2D (n=5 per group). Similarly, indexed left ventricular mass 
was already abnormal in 13 adolescent children with T2D compared to13 
obese controls (59). Table 2 demonstrates a mild improvement in E:A 
ratios with exercise training that does not return to healthy norms. Recent 
unpublished work using the speckle tracking methods proposed reveal 
additional abnormalities not detected by traditional echocardiography 
Interpretation of cardiac findings: These findings suggest that cardiac 
function is abnormal in relatively recently diagnosed and otherwise healthy 
children and adults with T2D during exercise even in the absence of clinically 
evident occlusive coronary artery disease and that this abnormality is related 
to impaired exercise capacity. The component of these early cardiac 
abnormalities in T2D related to decreased perfusion could be responsive to 
sitagliptin. 
 
2.0b Endothelial Dysfunction correlates with exercise impairment in T2D: 
We and others have reported that T2D is associated with impaired endothelial 
function (53, 65, 76, 89).We measured endothelial function using brachial 
artery ultrasound in 10 premenopausal women with type 2 diabetic and 10 
healthy women. T2D participants had smaller post-ischemic hyperemic 
increases in both brachial artery diameter and forearm blood flow than 
controls. Participants with T2D had lesser hyperemic brachial artery dilator 
(change in diabetics=0.028 ± 0.006 cm vs change in control =0.056 ± 0.008 
Control
watts
-50
0
50
100
150
200
PWCP (mmHg)
0
5
10
15
20
25
30
35
watts
-50
0
50
100
150
200
PCWP (mmHg)
0
5
10
15
20
25
30
35
Type 2 DM
 
Table 1. 
Controls (N=10) 
T2DM (N=10) 
Age 
39.3±6.6 
42.5±6.3 
Body Mass Index (kg/m2) 
28.3±3.9 
31.9±4.3 
HbA1C (%) 
5.0±0.4 
6.9±2.3* 
VO2 max (ml/kg/min) 
22.3±4.2 
18.7±2.3* 
Peak Respiratory Exchange Ratio 
1.14±0.05 
1.18±0.05 
Peak Cardiac Output (Fick) (L/min) 
13.1±2.8 
12.6±1.6 
Peak Cardiac Index (Fick) 
7.3±1.4 
6.8±0.5 
Peak A-V oxygen difference 
12.8±2.2 
12.3±1.3 
Peak Pulmonary Capillary Wedge 
Pressure (mmHg) 
16.7±3.7 
23.6±3.9* 
 VO2 max= maximal oxygen consumption; HR, Heart rate; A-V, arteriovenous *P<0.05 difference between  
 Controls and T2DM; +P=0.06 difference between controls and T2DM. Cardiac index=cardiac output/body 
 surface area; Max, maximal; Data are given as mean±SD  (11a,b- paper under review) 
Figure 3. Pulmonary capillary wedge 
pressure during graded exercise to VO2peak 
(77) 


Protocol Template 
Page 7 
CF-146, Effective 7/10/11 
 
cm, p<0.05) and hyperemic blood flow (change in diabetics= 16 ml 100 ml/ min vs change in control= 
26 ml 100 ml/ min, p<0.05) responses. 
Is there a relationship between flow mediated dilation and exercise capacity in T2D? Using data 
from a subsequent study (data analysis in process), we performed a correlation on the relationship 
between VO2peak and change in FMD from rest to hyperemic flow in persons with T2D.  The correlation 
was r=0.65, P<0.05, suggesting a relationship between endothelial function and exercise capacity in this 
group.  Endothelial function is abnormal in T2D in association with exercise abnormalities and 
improved by sitagliptin. 
 
2.0c Skeletal muscle oxygen delivery is slowed in T2D.  
Cardiac perfusion and function during exercise in T2D are 
abnormal but cannot account for all of the exercise impairment 
in T2D. One key factor associated with the exercise intolerance 
in T2D is abnormally slowed pulmonary VO2 kinetics during 
submaximal exercise as we have previously demonstrated 
(10,75).  The mechanisms of this maladaptation during 
exercise are unclear but likely relate to impairments in skeletal 
muscle blood flow.  We conducted a study to compare skeletal 
muscle deoxygenation ([HHb]) responses (indicating local 
muscle O2 extraction) and estimated microvascular blood 
flow (Qm) kinetics in T2D and healthy subjects following the 
onset of moderate exercise (4).  Pulmonary VO2 kinetics and 
[HHb] (using near infrared spectroscopy) were measured in 
11 T2D and 11 healthy subjects during exercise transitions 
from unloaded to moderate cycling exercise.  The increase 
in Qm were calculated using VO2 kinetics and [HHb] 
responses via rearrangement of the Fick principle.   We found 
that VO2 kinetics were slowed in T2D compared with controls 
(43.8±9.6s vs. 34.2±8.2s, P<0.05), and the initial [HHb] 
response following the onset of exercise transiently 
exceeded the steady state level of local muscle O2 extraction 
in T2D compared with controls.  This response was 
consistent with a greater dependence on O2 extraction early 
in exercise that was not observed in healthy muscle.  
Concordantly, the mean response time of Qm increase was 
prolonged in T2D compared with healthy subjects 
(47.7±14.3s vs. 35.8±10.7s, P<0.05).  Thus, skeletal muscle 
in T2D demonstrated a transient imbalance of muscle O2 
delivery relative to O2 uptake following exercise onset, suggesting a slowed Qm increase in T2D 
muscle.  This response could not be explained by a central defect in cardiac output as heart rate 
responses to this low level of exercise were similar between T2D and controls indicating the observed 
muscle blood flow response were likely related to events occurring local to the exercising muscle.  
Thus, impaired vasodilatation secondary to vascular dysfunction in T2D during exercise may 
contribute to this observation, potentially linking this finding to impaired vascular reactivity and 
endothelial dysfunction.  These data support the hypothesis that muscle blood flow kinetics (and 
hence O2 delivery) following the onset of exercise are impaired in T2D; a response that could be 
improved with sitagliptin.  
 
2.1 Studies demonstrating the ability of our laboratory to augment exercise function in T2D: 
 
0
60
120
180
240
300
360
[HHb] (% response) 
-20
0
20
40
60
80
100
120
140
0
60
120
180
240
300
360
[HHb] (% Response)
-20
0
20
40
60
80
100
120
140
CONTROL
T2DM
Overshoot
Figure 4.  Muscle deoxyhemoglobin ([HHb]) 
concentration kinetics in a representative healthy 
subject and a subject with T2D at rest and following 
the onset of moderate exercise.  Exercise begins at 
time = 0.  Note pronounced overshoot of HHb in the 
T2D subject early in the exercise transition.  This 
response indicates a greater reliance upon local 
muscle oxygen extraction (e.g. due to slowed 
increase in microvascular blood flow) early in 
exercise that is not observed in healthy muscle. (4) 


Protocol Template 
Page 8 
CF-146, Effective 7/10/11 
 
2.1a Does exercise training improve CV exercise performance in T2D?  Men and women with T2D 
and nondiabetic controls entered into a three month program of exercise training following baseline 
measures to assess effects of exercise training on endothelial and cardiac function (10) and in 
preparation for publication).  Participants had 36 sessions of exercise training.  During each training 
session, heart rate was kept between 70-85% of bicycle-ergometer measured VO2peak with work load 
being increased regularly to ensure that the training effect was continued. Resting measures of cardiac 
function (determined by echocardiography and tissue Doppler) made before exercise training 
revealed that both women and men with T2D had significant impairment in cardiac function. Exercise 
training improved exercise capacity, metabolic control, endothelial function and cardiac function but 
without normalizing any of these variables. Other studies have also described improvements in 
exercise capacity in T2D with exercise training (23,79,80) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Role of VO2 kinetics. Previously, we have demonstrated slowed pulmonary VO2 kinetic time 
constants in uncomplicated T2D compared with nondiabetics (10,75).  The magnitude of slowed VO2 
kinetics in T2D was correlated with the functional impairment in VO2peak.  We have also shown that 
exercise training significantly increases VO2peak and improves VO2 kinetics (e.g. faster time 
constants) in women with T2D but this does not fully normalize the exercise defect (10).  Because 
we observed an attenuated oxygen pulse response with exercise in T2D in the absence of treatment, 
the slowed VO2 kinetics may be attributed to a central cardiac defect in O2 delivery which could 
potentially be improved by sitagliptin.   
 
2.1b Can a pharmacological intervention increase VO2peak in people with T2D? 
Based on our observation that defects in functional exercise capacity correlated with decreased insulin 
sensitivity and decreased endothelial function, we hypothesized that a thiazolidinedione would improve 
exercise function (74).  20 patients with uncomplicated T2D were randomized in a double-blinded study 
to receive either 4 mg/day of rosiglitazone or matching placebo after baseline measurements which 
included endothelial function (by brachial ultrasound), VO2peak and insulin sensitivity with reassessment 
after 4 months of treatment.  The two groups of patients did not differ at baseline in any measure. 
Patients treated with rosiglitazone had significantly improved VO2peak (by 7%)(Table 3) as well as 
improved brachial artery diameter (BAD) and insulin sensitivity (74). Rosiglitazone increased the BAD 
response to cuff inflation by 83% over the baseline response (from 0.024±0.03 cm to 0.044±0.03 cm, 
P<0.05) while no significant increase seen in the response of placebo-treated controls (from 
0.010±0.02 cm to 0.017±0.02 cm, P=NS).  Change in VO2peak correlated with changes in BAD and 
insulin resistance in the rosiglitazone treated group only.  These data demonstrate that an agent that 
improves insulin sensitivity can improve exercise function in T2D.   
 
Table 3. Rosiglitazone Effects on Exercise Capacity in T2D  (*P<0.05 difference within group) 
 
  Placebo  (N=10) 
RosiglitazoneTreated 
(N=10) 
Table 2: Effects of exercise training on exercise capacity, endothelial function and diastolic function in  
T2D 
 
 
 
VO2peak 
ml/kg/min 
 
Respiratory 
exchange 
ratio 
Flow 
mediated 
dilation (Δ) 
Tissue 
Doppler 
E:A Lateral 
Tissue  
Doppler 
E:A 
Septal 
                Women   Pre 
                              Post 
18.6±2.3 
20.4±4.3* 
1.17±0.07 
1.18±0.11 
0.17±0.06 
0.27±0.23* 
1.09±0.20 
1.18±0.41+ 
0.99±0.22 
1.08±0.36 
                 Men       Pre  
                              Post    
25.0±6.0 
28.0±5.3* 
1.22±0.10 
1.22±0.10 
0.17±0.14 
0.28±0.16+* 
1.19±0.32 
1.25±0.27+ 
1.14±0.36 
1.08±0.36 
 Pre, Pre exercise training; Post, Post exercise training; VO2peak, maximal VO2; RER, respiratory exchange,  
  ** P<0.05 difference pre and post exercise training; +tendency towards difference pre and post exercise  
 training (9,23) 


Protocol Template 
Page 9 
CF-146, Effective 7/10/11 
 
VO2peak (ml/kg/min) 
                     Pre    
                     Post 
 
    19.4±5.2 
    18.1±5.3 
 
    19.8±5.3 
    21.2±5.1* 
Oxygen 
pulse 
(VO2/HR) 
                    Pre 
                    Post            
 
   11.3±3.7 
   11.1±3.6 
 
   13.3±3.7 
   14.8±4.1* 
 
2.2 Mitochondrial function-Mitochondrial respiration using MRS techniques in human 
subject correlates with functional exercise capacity (a pilot study from our lab):   Following 
a controlled study diet, in 11 adolescents (14.8±2.2 years), including n=5 T1D, n=1 T2D, n=3 lean 
and n=2 obese controls, were assessed in the fasting state for VO2peak via a bicycle exercise test, 
lean body weight (kg) via DEXA, and metabolite recovery time (phosphocreatine (PCr), inorganic 
phosphate (Pi), and ADP) via 
31P magnetic resonance spectroscopy. The soleus and 
gastrocnemius muscles were studied during isolated static plantar flexion at 70% maximum 
contraction. Measurements of PCr, Pi, and ADP were made at rest, 
during 90 seconds of contraction, and for 15 minutes following the 
exercise bout. A significant negative correlation was found between 
VO2peak (43.3±7.4 mL/min/kg) and post-exercise Pi recovery half 
time (23.9±6.4 secs) (r=-0.625, p=0.04).  A significant negative 
correlation was found between VO2peak (29.7±8.3 mL/kg/min) 
and post-exercise ADP recovery half time (17.8±5.1 secs) (r=-
0.644, p=0.03). Thus, metabolic recovery, regarding ADP and 
Pi recovery time, was slower in adolescents with decreased 
exercise capacity as determined by VO2peak.  Impaired ATP and 
PCr re-synthesis could minimize energy production and thereby 
reduce exercise capacity. We anticipate that sitagliptin will 
improve mitochondrial function by enhancing substrate delivery.    
 
3.0 What is the possible role of Sitagliptin? 
3.0a Can DPP-4 inhibitors improve exercise capacity?: No 
previous reports have investigated the impact of DPP-4 
inhibitors on exercise capacity in human subjects. However, the 
impact of GLP-1 and DPP-4 inhibitor on endothelial function, capillary recruitment, and eNOs activity 
would be predicted to improve exercise function by correcting the exercise defects we have identified. 
In human subjects, GLP-1 improves insulin dependent increases in forearm blood flow (86). In a 
study that parallels our work on kinetics, GLP-1 increased glucose clearance into the myocardium in 
subjects with T2D via early increases in myocardial blood flow (25). 
     Rodent studies demonstrate that sitagliptin improves endothelial function through eNOS in a GLP-
1 dependent fashion in the renal arteries (46) and recruits capillaries to increase microvascular 
endothelial surface area thereby improving insulin delivery and action in an NO dependent fashion 
(18). In other studies, sitagliptin improved angiogenesis in ischemic limbs by enhancing the function 
of endothelial progenitor cells and stimulation of eNOS  (36). We have demonstrated that the diabetic 
vasculature fails to induce eNOS or mitochondrial biogenesis in diabetic rats  (41). Saxagliptin 
restores the induction of eNOS and vascular mitochondrial protein expression in response to an 8 
day exercise exposure.  Taken together, the background and preliminary data support the plausibility 
of the hypothesis that sitagliptin will improve cardiac and muscle blood flow and increase both VO2 
kinetics and VO2 peak in people with T2D.  
 
3.0b. Effects of sitaglipin on mitochondrial function. As noted above, in GK rats, we recently 
showed that the response of eNOS, SIRT3, pCREB or mitochondrial protein expression with exercise 
in diabetes is impaired and that intervention with saxagliptin increased these markers as well as the 
0.000
5.000
10.000
15.000
20.000
25.000
30.000
Lean Control
Type 1
Obese
Type 2
1/2 Time for ADP depletion after 70% max effort exercise
N=12
N=4
N=8
N=8
Green and Nadeau unpublished
Seconds
Figure 5. Half-Time for ADP 
Depletion After 70% Max Effort 
During Exercise 


Protocol Template 
Page 10 
CF-146, Effective 7/10/11 
 
mitochondrial response to exercise in diabetes, restoring normal mitochondrial adaptation to exercise in 
a diabetes model (41). This is likely the result of the prevention of GLP-1 degradation by saxaglipitin and 
enhanced activation of vascular GLP-1 receptors. Therefore, we propose to evaluate the effect of 
sitagliptin on mitochondrial function in humans in the present study. 
 
Overall Interpretation: This body of evidence suggests that impairments in metabolic, endothelial, 
cardiac, peripheral circulatory function and mitochondrial function may each contribute to a portion 
(but not all) of the exercise defect in T2D. Thus, evaluation of the impact of sitagliptin on each of 
these factors contributing to the exercise impairment in diabetes is proposed.  
 
Summary and  Interpretation of Preliminary Data: Taken together, the above observations show 
that persons with T2D have substantially impaired exercise performance and identifies some key areas 
which may be targets for therapeutic intervention. It is notable that although all patients in the prior 
studies had uncomplicated diabetes and had only had disease for a relatively short period (<10 yrs), the 
functional impairment is very substantial.  Because the lower VO2peak has been associated with a 
reduced ability to carry out normal activities as well as excess mortality, any improvement of functional 
status is of great clinical significance. Endothelial dysfunction is present in T2D and also correlates 
with exercise impairment (76). Associated abnormalities of cardiac perfusion/function (77) and 
skeletal muscle oxygenation with exercise (4) suggest local abnormalities in O2 delivery within the 
exercising muscles (4). The proposed studies will examine the impact of sitagliptin on exercise 
capacity and cardiac and vascular function in T2D using a comprehensive assessment plan.   
 
Sitagliptin is a promising treatment for exercise abnormalities given its roles in improving cardiac, 
endothelial and mitochondrial function. Thus, this may be a powerful tool to get persons with T2D able 
to maximize their ability to exercise and thus improve their metabolic state and CV fitness.  
 
 
IV. Research Methods 
 
A.  Outcome Measure(s):   
Subjects: This study will randomize subjects with T2D into one of 2 groups (with 28 patients enrolled 
in total). The intervention will be: Group 1: sitagliptin 100 mg/d plus placebo. Group 2: glimepiride 2 
mg/day plus placebo. Patients on metformin monotherapy or those who are metformin-intolerant will 
be sought for this study (see inclusion and exclusion criteria). Drugs will dispensed by the University 
Research Outpatient Pharmacy. 
 
SA#1a. Overview of experimental design: We will compare exercise capacity in patients with T2D 
taking sitagliptin plus placebo vs patients on glimepiride plus placebo to determine whether a three 
month intervention with sitagliptin improves VO2peak and oxygen uptake kinetics.  
 
SA#1b. Overview of experimental design: Experiments under this aim will determine the effect of 
sitagliptin vs glimepiride upon established measures of cardiac function, endothelial function and 
peripheral circulatory function. Stress echocardiography with speckle tracking will be used to 
determine cardiac function. Endothelial function will be assessed by changes in brachial artery diameter 
in response to cuff occlusion. Vascular stiffness will be assessed using the Sphygmocor. Muscle tissue 
O2 saturation will be measured during exercise using near–infrared monitors. We will also assess the 
relationship between improvements in glycemic control and FFA and changes in exercise function. 
In addition to testing the stated hypothesis, this aim will also be hypothesis-generating in that the 
proposed studies will reveal which measures are improved most by sitagliptin.  This new information will 
help to design further grant proposals to the ADA and NIH to examine the cellular and molecular 
mechanisms by which sitagliptin has benefit for persons with T2D. 
 


Protocol Template 
Page 11 
CF-146, Effective 7/10/11 
 
SA#2: Overview of experimental design: Experiments under this aim will utilize MRS technology 
to determine whether sitagliptin normalizes mitochondrial function in human diabetic participants 
given sitagliptin plus placebo vs those given glimepiride plus placebo. 
 
B. Description of Population to be Enrolled:  
Twenty-eight sedentary persons with uncomplicated T2D between the ages of 22 and 70 will be 
randomized.  Subjects must be taking metformin only (500-2000 mg/day) or not on diabetes medication, 
if intolerant of metformin, to treat diabetes.  
Inclusion Criteria: 
1. Female subjects may be pre, peri or post-menopausal.   
2. People who do not participate in a regular exercise program (> one bout of exercise per week).   
3. Presence of T2D will be documented by chart review that will confirm the diagnosis as well as 
the presence of treatment for diabetes.   
4. Persons with T2D will be accepted for study only if they have total glycosylated hemoglobin 
levels (HbA1C) between 7.0 and 9.5% (adequate control) on therapy. 
5. Persons who are taking metformin 500-2000 mg/day only to control their T2D, but are not taking 
any other diabetes medication in addition to or instead of metformin. 
6. Persons not taking medication to control diabetes.  
 
Exclusion Criteria: 
1. Females of childbearing potential who are pregnant, planning to become pregnant or 
breastfeeding. 
2. Persons will be excluded if they have evidence of ischemic heart disease by history or abnormal 
resting or exercise electrocardiogram (EKG) (> 1 mm ST segment depression), regional wall 
motion abnormalities, LV systolic dysfunction or significant valvular disease.  
3. Persons with angina or any other cardiac or pulmonary symptoms potentially limiting exercise 
performance.   
4. Presence of systolic blood pressure >160 at rest or >250 with exercise or diastolic pressure >95 
at rest or >115 with exercise.   
5. Subjects who have peripheral arterial disease.  
6. Subjects with proteinuria (urine protein >200 mg/dl) or a creatinine > 2 mg/dl, suggestive of renal 
disease. 
7. Persons with liver function impairment defined as elevated liver function tests three times the 
upper limit. 
8. Persons with a history of pancreatitis. 
9. Subjects more than 140% of ideal body weight.    
10. Patients on insulin therapy will not be included. 
11. Current smokers will not be accepted for study since smoking can impair CV exercise 
performance but people who have quit smoking for at least 1year will be accepted for study. 
12. Persons with autonomic dysfunction (>20 mm fall in upright BP without a change in heart rate) 
will be excluded. 
13. Diabetic persons with clinically evident distal symmetrical neuropathy will be excluded from 
further study, because of possible effects on exercise performance, by evaluation of symptoms 
(numbness, paresthesia) and signs (elicited by vibration, pinprick, light touch, ankle jerks).   
14. Persons with diabetic ketoacidosis. 
15. Persons with a serious hypersensitivity to sitagliptin, sulfonylureas or sulfonamides. 
16. Inability to walk or ride a bike unassisted for a continuous 5 minutes. 
17. Subjects will be excluded if they have any implanted metal in their body. 
18. Subjects currently being treated with Digoxin. 
 
C. Study Design and Research Methods   


Protocol Template 
Page 12 
CF-146, Effective 7/10/11 
 
Study Protocol.  Subjects will come for a total of nine testing visits during which evaluations will take 
place.  Visits are structured as follows:  
1. After subjects review the study and give consent for study participation, a history and physical 
exam will be performed. Blood drawn for measurement of HbA1C, fasting glucose, fasting 
insulin, and microalbuminuria, CRP, IL-6, adiponectin, and creatinine. These measures will be 
covariates in the analyses.  Markers of inflammation including CRP, IL-6 and adiponectin are 
used to assess whether inflammation is present.  In this way, we can determine if exercise 
capacity is affected by markers of inflammation. The glucose and insulin are measured to 
assess glycemic status.  Additional screening labs include CBC, FSH, urine protein and a lipid 
panel to assess whether women are pre- or post-menopausal (FSH), and overall health (CBC, 
lipids and urine protein). If participants opt not to fast for visit 1, we will postpone the blood draw 
to visit 2. Ankle brachial index, ANS function tests, the LoPAR questionnaire and vital signs will 
be performed. 
2. A dietary survey will be administered for food preferences for the two day study diet 
administered prior to visits 3 and 7 and a study meal and snack prior to visits 4 and 8. DEXA 
and body composition tests will be done to ensure that groups are weight similar (using fat-free 
mass). A pulmonary function test, resting EKG and familiarization bicycle test will be performed.  
3. Subjects will be given an option of washing out from exclusionary medications or if their 
hemoglobin A1C results are below the threshold for inclusion. Specific washout periods for 
medications are as follows:  
a. Metformin – 8 week washout.  
b. Thiazoladinediones (e.g., pioglitazone, rosiglitazone) – 8 week washout 
c. DPP-4 inhibitors (e.g., sitagliptin, saxagliptin, etc.) – 30 day washout 
d. Sulfonylureas (e.g., glipizide, glyburide, glimepiride) – 30 day washout 
If subjects elect to perform a medication washout, approval from their primary care physician 
will be obtained and they will be closely monitored throughout the washout period and 
continuing until they are randomized to the study medication. 
4. Subjects will receive a two day study diet prior to visit 3. A resting and exercise EKG will be 
performed on the day of the visit. A graded exercise test will be done to determine the VO2peak.  
Patients will have measures of cardiac function and endothelial function on visit 3 by 
plethysmography and cardiac echo. Vital signs and arterial stiffness/endothelial function will 
be non-invasively measured by the Sphygmocor system will be taken at rest. If the subject 
participated in a medication washout, fasting glucose and HbA1C screening labs will be 
repeated. 
5. Subjects will receive a study meal and snack prior to visit 4. Calf muscle MRS will be performed 
on a 3.0 T whole–body MRI scanner.  
6. During visit 5, subjects will perform three constant-load tests to measure VO2 kinetics where 
StO2 will be measured during exercise. A resting and exercise EKG and vital signs will be 
performed during the visit. Subjects will be randomized to taking sitagliptin plus placebo or 
glimepiride plus placebo and all must be taking metformin (500-2000 mg /d) for 3 months 
(except those who are metformin-intolerant). Sitagliptin and its placebo will be administered 100 
mg/d. Glimepiride and its placebo will be administered 2 mg/day. During the treatment phase 
subjects will be given a log to keep track of their blood glucose each day.  Study coordinators 
will contact each subject weekly to obtain these values which will be checked by the study 
doctors and shared with the subject’s primary care physician if adjustments in other medications 
need to be made. 
7. Visit 6 will consist of a physical exam with a clinician as well as a blood draw and check of vital 
signs during sitagliptin or glimepiride treatment.   
8. After 3 months of sitagliptin or glimepiride administration, Visit 3 will be repeated. Additional 
testing to be performed during visit 7 will include a physical exam performed by a study 
physician, blood work for covariate lab tests listed in Visit 2 and the LoPAR questionnaire.  
9. During visit 8, visit 4 procedures will be repeated. 


Protocol Template 
Page 13 
CF-146, Effective 7/10/11 
 
10. During visit 9, the testing performed during visit 5 will be repeated.  


Protocol Template 
Page 14 
CF-146, Effective 7/10/11 
 
Visit Schedule:  
Visit Timeline 
Screening 
Visit 1 
Screening 
Visit 2 
Visit 
3 
Visit 
4 
Randomization 
Visit 5 
Visit 
6 
Week 
2 
Visit 
7 
Week 
12 
Visit 
8 
Week 
13 
Visit 
9 
Week 
14 
Informed Consent and HIPPA 
Auth. 
X 
  
  
  
  
  
  
  
  
History & Physical  
X 
  
  
  
  
X 
X 
  
  
LoPAR 
X 
  
  
  
  
  
X 
  
  
ANS Function Tests 
X 
  
  
  
  
  
  
  
  
Ankle Brachial Index 
X 
  
  
  
  
  
  
  
  
Pulmonary Function Test 
  
X 
  
  
  
  
  
  
  
Blood Draw and Urinalysis 
X  
 
  
  
  
X 
X 
  
  
Vital Signs 
  
X 
X 
  
X 
X 
X 
  
X 
Electrocardiogram 
  
X 
X 
  
X 
X 
X 
  
X 
Plethysmography, Cardiac 
Echo, FMD 
  
  
X 
  
  
  
X 
  
  
DEXA 
  
X 
  
  
  
  
  
  
  
GXT 
  
X 
X 
  
  
  
X 
  
  
Calf MRS 
  
  
  
X 
  
  
  
X 
  
Kinetics 
  
  
  
  
X 
  
  
  
X 
Near-infrared spectroscopy 
  
  
  
  
X 
  
  
  
X 
Diet Instruction 
  
X 
  
  
  
  
  
  
  
Study diet 
  
  
X 
X 
 
  
X 
X 
 
Arterial Stiffness 
Measurement 
  
  
X  
  
 
  
 X 
  
 
Study drug dispension 
  
  
  
  
X 
  
  
  
  
Length of Visit 
2.5 hrs 
2.5 hrs 
2.5 
hrs 
1.5 
hrs 
2.5 hrs 
1 hr 
3 hrs 
1.5 
hrs 
2hrs 
 
D. Description, Risks and Justification of Procedures and Data Collection Tools: 
Procedures: 
Body Composition and DEXA.  These measures will be performed according to standard methods 
(47). 
Dietary Interviews. Customary macronutrient pattern will be ascertained by food preferences 
questionnaire.  A study diet provided by the clinic will be used prior to certain visits as stipulated in 
the Visit schedule. The clinic dietician will prescribe a standardized nutrient breakdown including 
20% protein, 35% fat and 45% carbohydrate for the two days prior to visits 3 and 7 and stud meal 
and snack prior to visit 4 and 8. The range of sodium in the CTRC diet will be about <2300 mg/day. 
In addition, sodium intake guidelines will be followed throughout the study to assure standard 
hydration and especially with visits where the CTRC diet will be utilized for diets prior to the visits. 
Brachial Artery Diameter.  Endothelial function as assessed by the brachial artery method will be 
utilized following the protocol described by Celermajer et al.(14, 15, 85) that measures dilation of the 
brachial artery by ultrasound in response to hyperemia.  The brachial artery diameter will be 
measured using B-mode ultrasound images, with the use of a GE Vivid 7 ultrasound system and a 


Protocol Template 
Page 15 
CF-146, Effective 7/10/11 
 
10.0-mHz linear-array transducer as previously reported in our laboratory (GE vivid 7 Dimension, 
Milwaukee WI.). Scans will be obtained with the subject at rest and following reactive hyperemia.  
Low Level Physical Activity Recall Questionnaire (LoPAR): This questionnaire has been 
validated for use in persons with T2DM and peripheral arterial disease as well as in sedentary 
controls by the Investigator as previously reported and is being used as an outcome measure by 
Diabetes Prevention Program (44).The LOPAR asks about physical activity level over the previous 
week.  Subjects are asked in a series of questions to itemize their time (reporting specific activities) 
into work, leisure and housework categories.  Questionnaire results are calculated in metabolic 
equivalents (METS) where one MET equals resting VO2 (3.5 ml/kg/min).  This questionnaire will be 
used in this study as a screen to ensure that all participants are sedentary.  
Skeletal Muscle Oxygenation. (Near-infrared Spectroscopy) Skeletal muscle [HHb] will be 
assessed by a frequency domain multi-distance NIRS monitor (Optiplex TS, ISS, Champaign, IL, 
USA) during each CWR exercise test (40).  The use and limitations of NIRS have been extensively 
reviewed (4, 24, 27). The NIRS monitor uses two wavelengths of NIR light (690 and 830nm) and four 
light source-detector distances at 2.0, 2.5, 3.0 and 3.5 cm.  Local muscle O2 extraction was 
determined as the change in [HHb] as previously described (5, 10, 42). The NIR data will be sampled 
continuously at rest and during exercise and recorded at 50Hz.  The device probe will be positioned 
on the distal third of the vastus lateralis of the dominant limb, secured using a Velcro strap, and 
covered with a cloth bandage to exclude ambient light.  The NIRS monitor is calibrated prior to each 
visit using a calibration phantom of known scattering and optical properties.    
Plethysmographic Measurements.  Forearm blood flow will be determined in seated participants 
by venous occlusion strain gauge plethysmography (D.E. Hokanson Inc. Issaquah, WA), using 
calibrated mercury-in-silastic strain gauges and expressed as ml/100 ml/min (3, 33, 34).  The arm 
will be supported above the heart level.  Venous occlusion pressure will average 35 mmHg.  
Circulation to the hand will be prevented by inflating a wrist cuff to suprasystolic pressure before 
each forearm blood flow determination.  Determination of forearm blood flow will comprise at least 
five separate measurements performed at 10-15 sec intervals. After a 10-15 min rest period, 
measurements will be repeated (3-5 times). 
Cardiac 
Echocardiographic 
Measurements. 
Standard 
two 
dimensional 
and 
Doppler 
echocardiography will be performed using standard methods (32, 73) to exclude the presence of left 
ventricular systolic dysfunction, regional wall motion abnormalities (suggestive of coronary artery 
disease), pericardial disease or significant valvular pathology (GE Vivid 7 Dimension, Milwaukee, WI).  
Chamber sizes, LV end-systolic and diastolic chamber dimensions and wall thickness, fractional 
shortening and the area-length method for measurement of cardiac volume in order to measure ejection 
fraction will be quantitated by standard techniques for all individuals.  
Cardiac function will be assessed by standard echocardiographic parameters (E and A wave 
velocities, E:A ratio, E wave deceleration time, pulmonary vein Doppler) as well as tissue Doppler of 
both the septal and lateral mitral annuli (E' and A' velocities, E':A' ratio, E:E' ratio) and flow 
propagation determined by color M-mode (32,73). Measurements will be collected at rest and post-
exercise (supine). Dr. Jennifer Dorosz , MD, Collaborator who has expertise in echocardiography and 
these techniques, will be blinded to the diagnostic status of the participant and will supervise acquisition 
of all of the echocardiographic data as well as perform all of the measurements and interpretation.  
Echocardiograms will be obtained with a commercially available ultrasound system (GE Vivid 7 
Dimension, Milwaukee, WI).  Subjects will be examined in the left lateral decubitus position using 
standard parasternal, short-axis, and apical views. All recordings and measurements will be obtained 
by the same observer according to the recommendations of the American Society of 
Echocardiography  and will always be performed at the same time of day for each subject to avoid 
the possible influence of circadian rhythm on left ventricular diastolic function.  No subject will have 
echocardiographically detectable regional wall motion abnormalities, and each subject will have 
normal left ventricular systolic function. All cardiac valves will be examined to rule out significant 
valvular disease. Left ventricular mass (LVM) will be calculated using the following formula: LVM (g) 
= 0.8 x 1.04 [(LVEDD + IVST + PWT)3 - (LVEDD)3] + 0.6, where LVEDD is left ventricle end diastolic 


Protocol Template 
Page 16 
CF-146, Effective 7/10/11 
 
internal diameter, IVST is interventricular septal thickness, and PWT is left ventricular posterior wall 
thickness. Measurements made during exercise will include the ratio of mitral inflow E over the ratio 
of E' from tissue Doppler (29). 
Calf MRS. (94) Subjects will be fasting and refrain from physical activity for 3 days prior to the study. 
Calf muscle MRS will be performed as described in preliminary studies on a 3.0 T whole–body MRI 
scanner (GE Medical Systems, Waukesha, WI), using a dual frequency 31P/1H Flex Coil (Clinical MR 
Solutions). Scout images of the dominant leg will allow determination of the widest gastrocnemious 
and soleus cross sectional area. A resistance strap will be affixed to the hips, dominant leg, ankle 
and foot, on a specialized portable, in-MRI exercise bench to allow isotonic plantar flexion against 
force. Contractions will be performed at 30%  and 60%  of the subject’s MVC following the calf 
exercise protocol described below (single leg, plantar flexion exercise tests) monitored by a Single 
Point Load Cell (Tedea Model 1250), connected via an A/D board to a laptop for analysis on LabView 
software. After scout images and shimming using the 1H portion of the flex coil, 31P measurements 
(phosphocreatine, free Pi and ATP peaks are measured while ADP and pH are calculated from 
JmRUI algorithms) will be made at rest, during calf exercise for five minutes, and for at least 15 min 
immediately following the exercise bout. Total study time will be 1.5 hours. Phosphocreatine recovery 
time will be analyzed to determine the rate of oxidative ATP synthesis from ADP using JmRUI (48). 
Graded Exercise Test.  To determine the VO2peak and lactate threshold, in all persons, participants, a 
graded bicycle protocol to exhaustion will be carried out as previously described (10,75,78).   
Constant load exercise test.  To make VO2 kinetic measurements, constant load bicycling will 
performed as previously described at low- to moderate-work-loads at approximately 85% of the lactate 
threshold (steady state) (10,75,78).   
Oxygen Uptake Kinetic Measurements.  Using the breath by breath capabilities of our metabolic cart, 
we will measure kinetics of VO2 below and above the lactate threshold as we have done in prior studies 
according to published methods (10,75,78).   
Arterial Stiffness Measurement: The Sphygmocor Px/Vx (Actor Medical) will be used to non-
invasively assess arterial stiffness in the form of pulse wave velocity (PWV) and augmentation 
index (AI) during visits 3 and 7.  Three electrocardiography leads are placed with removable 
adhesive on the chest wall, for the purpose of timing systole.  A small metal tonometer is 
sequentially placed on the skin overlying the radial, femoral and carotid arteries.  The anterior wall 
of the artery is flattened, as with palpation of a pulse.  The tonometer non-invasively measures the 
pressure pulse waveform at each of these sites and correlates this with electrocardiographic data 
and the distance between the femoral artery and carotid artery sites to determine pulse wave 
velocity.  The accuracy and reproducibility of the Sphygmocor Px/Vx have been validated, 
previously (78). 
Autonomic Nervous System Function Testing (ANS): Study participants will undergo supine 
resting electrocardiogram (ECG) recording for 5 minutes in visit 1 in a quiet room, with a stable 
room temperature. Participants will be asked to relax but not fall asleep and to breathe normally 
and remain still without talking during the ECG recording. Following the resting ECG, a second 
supine ECG will be recorded while study participants take in a deep breath for 10s. The ECG 
recordings will be transmitted electronically to the Epidemiological Cardiology Research Center 
(EPICARE) Center (Wake Forest University School of Medicine, Winston-Salem, NC) for analysis, 
interpretation, and calculation of heart rate variability. The SD of consecutive RR intervals of the 
entire record in Lead II will be used as a measure of overall HRV. In addition, autoregressive power 
spectral analysis will be used to determine the spectral power in the following bands: high 
frequency (HF, 0.15–0.45 Hz, reflects parasympathetic outflow), low frequency (LF, 0.04–0.15 Hz, 
reflects both sympathetic and parasympathetic outflow), and very low frequency (0.01–0.04 Hz). 
LF/HF and HF normalized to total power will be analyzed as measures of sympathetic and 
parasympathetic outflow, respectively. 
Randomization: Twenty-eight subjects will be randomized to blinded treatment of sitagliptin 100 
mg plus placebo or glimepiride 2 mg plus placebo. This is a 1:1 randomization ratio. The UCH 
research pharmacy will provide the randomization schema as well as the blinded study medication 
for each subject. To ensure that our groups are similar for age and sex, we are very careful in our 
recruitment.  We have not found it necessary in any prior study to use a stratification scheme and 


Protocol Template 
Page 17 
CF-146, Effective 7/10/11 
 
we have always been able to successfully attain equal distribution in our groups. We have imposed 
exclusion criteria that will aid with this process. 
Sitagliptin Administration.  Participants randomized to the sitagliptin treatment arm will receive 100 
mg/d sitagliptin and 2mg/d placebo for 3 months. 
Glimepiride Administration. Patients randomized to the glimepiride treatment arm will receive 2 
mg/d glimepiride and 100mg/d placebo for 3 months. 
Blood and Urine Collection and Preparation.  Samples will be collected at baseline for the 
measurement of glucose, insulin, HbA1C, FSH, creatinine, adiponectin, CBC, urine protein, lipids, CRP, 
IL-6, and microalbumin. For females of childbearing potential, a pregnancy test will be performed at 
visits 2,5, 6 & 7. Samples will be collected after two weeks of treatment and after three months of 
treatment for the measurement of glucose, insulin, HbA1C, creatinine, CBC, urine protein, CRP, IL-6, 
adiponectin, and microalbumin. 
Monitoring for Study Drug Side Effects.  The most common side effects of sitagliptin and glimepiride 
include hypoglycemia and gastrointestinal symptoms. The rare but most severe side effects include eye 
issues, pancreatitis, and renal impairment. We will be monitoring for all of these potential side effects in 
the following ways:  
1. People who have a history of pancreatitis or kidney disease will be excluded from the study. 
2. Subjects will be instructed to check and log their blood glucose twice a day during the treatment 
period.  
3. Subjects will have access to the study team 24/7 via pager.  
4. Two to three days after a participant begins the study medication, the study PRA will follow-up by 
phone call to assess whether the subject is experiencing any symptoms.  At that time, the study PRA 
will also review the participant's blood glucose readings to date and advise the study physician if any 
readings are below 90 (see paragraph below). 
5. Two weeks after a participant begins the study medication, subjects will return to the clinic for a 
physical exam and blood draw. Blood glucose log will be reviewed and symptoms of study drug side 
effects will be assessed.  
6. The remaining time that subjects are on study drug, the research team will contact subjects weekly to 
assess possible symptoms.  
7. If symptoms are noted, study investigators will assess and treat symptoms appropriately. Subjects 
will be withdrawn from the study if the pharmacological agents prove harmful to them. 
 
Blood glucose will be monitored at every visit. While the subjects are taking study drug, we will ask them 
to monitor their blood glucose daily. To ensure subjects can monitor their glucose properly, subjects will 
be instructed during their randomization visit on proper finger stick blood glucose measuring technique.  
After randomization, subjects will be asked to monitor blood glucose twice daily; this will include daily 
monitoring of fasting blood glucose as well as an additional check at varying times (i.e. alternating before 
lunch, dinner, and before bedtime).  Subjects will also be instructed to check their blood glucose PRN if 
they experience any symptoms of hypoglycemia – these potential hypoglycemia symptoms will be 
written out and verbally explained to patients.  The study team will review these values weekly. Repeated 
measurements of blood glucose <90 mg/dl will be reported to the study staff so that a determination can 
be made on whether study medication dose adjustments are warranted to prevent hypoglycemia.  Any 
measurement of blood glucose <70 mg/dl will result in study investigators determining whether the 
subject needs to be withdrawn from the study or prescribed medication dosages to prevent recurrent 
hypoglycemia. 
Time-line. It is estimated that this study will take two years to complete. Fourteen persons per year will 
be studied. 
Recruitment of Patients. Patients will be recruited by newspaper and radio advertising to the 
community, by University-wide email recruitment announcements and from the internal medicine and 
diabetes clinics at the University of Colorado Denver School of Medicine.  Dr. Reusch sees patients with 
T2D and in addition, Drs. Reusch and Regensteiner are frequent speakers at local events targeted to 
persons with diabetes and will recruit patients at these events. 


Protocol Template 
Page 18 
CF-146, Effective 7/10/11 
 
 
Risks: 
Sitagliptin. 
Pancreatitis There have been postmarketing reports of acute pancreatitis, including fatal and non-
fatal hemorrhagic or necrotizing pancreatitis, in patients taking sitagliptin. After initiation of 
SITAGLIPTIN, patients should be observed carefully for signs and symptoms of pancreatitis. If 
pancreatitis is suspected, sitagliptin should promptly be discontinued and appropriate management 
should be initiated. It is unknown whether patients with a history of pancreatitis are at increased risk 
for the development of pancreatitis while using sitagliptin.  
Renal Impairment: Assessment of renal function is recommended prior to initiating sitagliptin and 
periodically thereafter. A dosage adjustment is recommended in patients with moderate or severe 
renal insufficiency and in patients with ESRD requiring hemodialysis or peritoneal dialysis. Caution 
should be used to ensure that the correct dose of sitagliptin is prescribed for patients with moderate 
(creatinine clearance ≥30 to <50 mL/min) or severe (creatinine clearance <30 mL/min) renal 
impairment.  
There have been postmarketing reports of worsening renal function, including acute renal failure, 
sometimes requiring dialysis. A subset of these reports involved patients with renal insufficiency, 
some of whom were prescribed inappropriate doses of sitagliptin. A return to baseline levels of renal 
insufficiency has been observed with supportive treatment and discontinuation of potentially 
causative agents. Consideration can be given to cautiously reinitiating sitagliptin if another etiology 
is deemed likely to have precipitated the acute worsening of renal function. Sitagliptin has not been 
found to be nephrotoxic in preclinical studies at clinically relevant doses, or in clinical trials.  
Use with Medications Known to Cause Hypoglycemia:  When sitagliptin was used in combination 
with a sulfonylurea or with insulin, medications known to cause hypoglycemia, the incidence of 
hypoglycemia was increased over that of placebo used in combination with a sulfonylurea or with 
insulin. Therefore, a lower dose of sulfonylurea or insulin may be required to reduce the risk of 
hypoglycemia.  
Hypersensitivity Reactions: There have been postmarketing reports of serious hypersensitivity 
reactions in patients treated with sitagliptin. These reactions include anaphylaxis, angioedema, and 
exfoliative skin conditions including Stevens-Johnson syndrome. Onset of these reactions occurred 
within the first 3 months after initiation of treatment with sitagliptin, with some reports occurring after 
the first dose. If a hypersensitivity reaction is suspected, discontinue sitagliptin, assess for other 
potential causes for the event, and institute alternative treatment for diabetes. 
Angioedema has also been reported with other dipeptidyl peptidase-4 (DPP-4) inhibitors. Use caution 
in a patient with a history of angioedema with another DPP-4 inhibitor because it is unknown whether 
such patients will be predisposed to angioedema with sitagliptin. 
Glimepiride.  
Hypoglycemia: As a result of the blood-sugar-lowering action of glimepiride, hypoglycemia may occur, 
and may also be prolonged. Possible symptoms of hypoglycemia include headache, ravenous hunger, 
nausea, vomiting, lassitude, sleepiness, disordered sleep, restlessness, aggressiveness, impaired 
concentration, alertness and reactions, depression, confusion, difficulty in speaking and even speech 
loss, visual disorders, tremor, pareses, sensory disturbances, dizziness, helplessness, loss of self-
control, delirium, cerebral convulsions, somnolence and loss of consciousness up to and including coma, 
shallow respiration and slow heart rate (bradycardia). In addition, signs of adrenergic counter-regulation 
may be present such as sweating, clammy skin, anxiety, rapid heart rate (tachycardia), hypertension, 
palpitations, angina pectoris, and cardiac arrhythmias.  
Eyes: Especially at the start of treatment, temporary visual impairment may occur due to the change in 
blood sugar levels. 
Digestive tract: Occasionally, gastrointestinal symptoms such as the following may occur: nausea, 
vomiting, sensations of pressure or fullness in the epigastrium, abdominal pain, and diarrhea. 
In rare cases, liver enzyme levels may increase. In isolated cases, impairment of liver function (e.g. with 
cholestasis and jaundice) and hepatitis may develop, possibly leading to liver failure. 


Protocol Template 
Page 19 
CF-146, Effective 7/10/11 
 
Blood: Severe changes in the blood picture may occur: Rarely, thrombopenia and, in isolated cases, 
leucopenia, hemolytic anemia or, e.g. erythrocytopenia, granulocytopenia, agranulocytosis, and 
pancytopenia (e.g. due to myelosuppression) may develop. 
Other adverse reactions: Occasionally, allergic or pseudoallergic reactions may occur, e.g. in the form 
of itching, urticaria or rashes. Such reactions may be mild, but also may become more serious and may 
be accompanied by dyspnea and a fall in blood pressure, sometimes progressing to shock. If urticaria 
occurs, a physician must be notified immediately. In isolated cases, a decrease in serum sodium, 
inflammation of blood vessels (allergic vasculitis) and hypersensitivity of the skin to light may occur. 
Pregnancy and lactation: To avoid risk of harm to the child, glimepiride must not be taken during 
pregnancy; a changeover to insulin is necessary. Patients planning a pregnancy must inform their 
physician, and should change over to insulin. Ingestion of glimepiride with the breast milk may harm the 
child. Therefore, glimepiride must not be taken by breast-feeding women, and a changeover to insulin 
or discontinuation of breast-feeding is necessary. 
Magnetic Resonance Sonography. The MRI is a non-invasive scan of your abdominal area and 
calf muscle, respectively, using a magnet. There is no radiation and no risk involved with the MRI. 
The MRI may be loud, therefore the subject is provided with audio protection and optional 
television to help increase comfort. Some subjects might feel claustrophobic while having an MRI 
and the scan will be stopped if it cannot be tolerated. In addition, any subjects with implanted metal 
cannot have an MRI due to the magnet involved. 
DEXA. During DEXA testing the amount of radiation received is less than 25mRem total body 
equivalent dose per measurement.  This is estimated to be less than one-half the amount received 
from a chest x-ray.  The radiation in this study is not expected to greatly increase these risks, but the 
exact increase in such risk is not known.  Women who are or could be pregnant should receive no 
unnecessary radiation, and should not participate in this study. 
Blood Draws. Pain when the needle goes into the vein.  A bruise may form at the site. 
Plethysmography/FMD (Blood flow measurements). There may be a slight discomfort while the 
arm cuff is inflated. 
GXT (Graded Exercise Test) and Kinetics. The exercise test is a standard procedure.  This test is 
done routinely with hundreds of heart patients every year.  The death rate of this test is approximately 
one in 10,000 tests.  The incidence of serious complications including prolonged arrhythmias (irregular 
heartbeats) or prolonged chest pain is approximately 4 in 10,000 tests.  This test will be performed on 
a Stationary bicycle. 
Arterial Stiffness Measurement. There is a very slight chance that fainting or stroke may occur.  No 
actual fainting or stroke has ever been reported.  The risk of these happening has been estimated to 
be less than 1/1,000,000. 
Medication washout: Risks of medication washout include high blood glucose levels, and in turn an 
increase of the hemoglobin A1C of the patient. 
 
    
E. Potential Scientific Problems:   
One potential pitfall is the use of sitagliptin since we have not evaluated its effects on exercise 
capacity.  However, although we have not used this agent before, others have demonstrated that 
GLP-1 can improve exercise capacity in some patient groups.  Given the finding that there are 
cardiac, endothelial and mitochondrial abnormalities associated with exercise impairment in T2D 
and the likely beneficial effects of sitagliptin on the heart, endothelial function and mitochondrial 
function in humans and animals, the proposed testing of this agent is both logical and innovative. 
Another potential pitfall is that we have not specifically evaluated the effect of metformin on 
exercise capacity. However, a recent report in healthy subject suggested that metformin did not 
improve exercise function; although this study was not in people with diabetes (11). In addition, in our 
prior studies, about 50% of patients have been on metformin and these patients’ results did not 
differ from the results of T2D patients not on metformin in terms of exercise capacity (or other 
measures of cardiac or endothelial function). Therefore we do not anticipate that metformin will 
have effects on exercise capacity in the present study. Other issues that will be investigated 


Protocol Template 
Page 20 
CF-146, Effective 7/10/11 
 
potentially in further studies include the possibility that a different GLP-1 agent may have even 
stronger benefits or that a different type of agent could have similar or better effects. 
 
F. Data Analysis Plan:   
Power Analysis  
Study Design: 
 
The proposed trial investigates the impact of sitagliptin on CV exercise capacity in individuals 
with T2D. Improved exercise capacity could reduce barriers to regular physical activity and improve CV 
health. We will enroll up to 75 study participants at the first visit. A total of 28 participants with diagnosed 
T2D will be randomized to receive either sitagliptin plus placebo or glimepiride plus placebo. The sample 
size was chosen so that the study had 94% power, even with about 15% dropout, and a reduction of the 
total sample size from 28 to 24.  If, in fact, the study randomizes 28 participants, and only 10 are left per 
group after dropout, for a total sample size of 20, the power of the study will still be high.  To detect a 
mean difference of 3.0 mL/kg3 in change in VO2 peak between the treatment groups, power  will be 
0.89 with a Type I error rate of 0.05, for 10 participants per treatment group.  Thus, the study should 
have adequate power even if there is a 28% dropout rate. 
 
Peak oxygen consumption (VO2peak) and oxygen uptake kinetics (VO2kinetics) will be 
measured prior to treatment and three months post-treatment. The primary hypothesis is that sitagliptin 
plus placebo will lead to greater improvement in functional exercise capacity in people with T2D when 
compared to glimepiride plus placebo. Secondary analyses will investigate the impact of sitagliptin plus 
placebo on cardiac function, endothelial function and tissue oxygen saturation (STO2) as well as health-
related quality of life. 
Proposed statistical analysis: We plan to fit a general linear univariate model with the change in VO2peak 
as the outcome, and treatment and baseline VO2peak as the predictors. We will test the null hypothesis 
of no difference between the treatment groups on change in VO2peak, using an F test. We will fit a 
similar model with VO2kinetics as the outcome. The power analysis is based on the primary hypothesis 
of interest: the drug-mediated change in VO2peak.  
 
To control Type I error rate for the experiment, a possible alpha-spending approach would be 
to test the hypothesis about VO2peak at a Type I error rate of 0.02, the hypothesis about VO2kinetics at 
0.02, and the secondary hypothesis at 0.01, for an experiment-wise error rate of 0.05. Because each 
hypothesis test is independently scientifically meaningful, we plan to test each hypothesis instead at an 
uncorrected Type I error rate of 0.05. The power analysis reflects this approach. 
Power Summary for Grant:  
 
We calculated unconditional power (101) for the F test of no difference in change from baseline 
for VO2peak between the two treatment groups, when controlling for baseline VO2peak. We assumed a 
standard deviation of 2 mL/kg for the change in VO2peak, a standard deviation of 5 mL/kg in baseline 
VO2peak, and a correlation of -0.17 between the baseline VO2peak and the change in VO2peak. 
Estimates were based on prior studies of the effect of rosiglitazone, a related drug, on exercise capacity 
in Type 2 diabetics.(2) The power analysis assumed a standard deviation of 2 mL/kg for the change in 
VO2 peak, a standard deviation of 5 mL/kg in baseline VO2 peak, and a correlation of -0.17 between 
the baseline VO2 peak and the change in VO2 peak. We expect the change in VO2peak to be at least 
3 mL/kg higher in patients taking sitagliptin plus placebo as compared to patients randomized to 
glimepiride plus placebo. This effect is clinically meaningful and of a magnitude similar to that observed 
in the study by Regensteiner et al.(74) 
 
To detect a mean difference of 3.0mL/kg (102) in change in VO2peak between the treatment 
groups, power will be 0.89 with a Type I error rate of 0.05, for 10 participants per treatment group. If we 
plan 12 participants per group, however, the power increases to 0.94. The modest increase in sample 
size leads to much higher power to detect the treatment effect. To ensure a conclusive study, we will 
target an effective sample size of 12 participants per group. Over the three months, we expect to lose 
up to two participants per treatment group. To account for the attrition, we have set an enrollment 
goal of 28 participants, or 14 participants per treatment group. 
 


Protocol Template 
Page 21 
CF-146, Effective 7/10/11 
 
Table 1. Power to detect a difference in change in VO2peak (Post–Pre) at a Type I error rate 
of 0.05 
Enrollment Goal 
Before Attrition 
Number of 
Participants Per 
Treatment 
Group After 
Attrition 
Total 
Participants 
After Attrition 
Detectable 
Difference(mL/kg) 
Power 
24 
10 
20 
3.0 
0.89 
26 
11 
22 
3.0 
0.92 
28 
12 
24 
3.0 
0.94 
 
Data Analysis. Comparisons between exercise performance and endothelial function in persons taking 
sitagliptin vs persons taking placebo will be made using unpaired t-tests.  Comparisons between key 
variables before and after treatment will be made within each group using paired t-tests. Multivariate 
regression techniques will be used to evaluate the influences of covariates.  Possible covariates 
(analyzed using ANCOVA) include but are not limited to insulin, fasting and post-prandial glucose and 
FSH levels.  Weight loss will be carefully controlled for since this is a possible result of sitagliptin 
administration.  Relationships between variables (i.e. insulin sensitivity and VO2peak) will be assessed 
using the Pearsons product moment correlation coefficient (r). We plan to fit a general linear univariate 
model with the change in VO2 peak as the outcome, and treatment and baseline VO2 peak as the 
predictors. We will test the null hypothesis of no difference between the treatment groups on change in 
VO2 peak, using an F test. We will fit a similar model with VO2kinetics as the outcome.  
 
We will handle dropout using case-wise deletion.  Because the power analysis shows that the study will 
have adequate power even with 28% dropout, this is a conservative approach. 
 
G. Summarize Knowledge to be Gained:   
T2D is associated with impairment in functional exercise capacity which may be caused by 
endothelial and/or cardiovascular dysfunction.  Sitagliptin works by inhibiting dipeptidyl peptidase 
4 (DPP-4) inactivation of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-
dependent insulinotropic polypeptide (GIP). This increases active incretin and insulin levels, and 
decreases glucagon levels and postglucose-load glucose excursion. Increasing evidence 
suggests that sitagliptin and other DPP-4 inhibitors may also provide protection against CV 
events (39,54,56), although the mechanism for this is not clearly understood. In addition, in a 
recent study from our lab, saxagliptin restored vascular eNOS and mitochondrial induction after 
an 8 day exercise intervention in a rodent model of diabetes (41).  
Incretins have additional relevant effects that are of particular interest for this proposal including 
benefiting endothelial and mitochondrial function (22,26), as well as reducing CV risk (51). As has 
been noted, GLP-1 is a critical factor for healthy metabolism and vasculature and stimulates 
eNOS (26), (22).  Small studies in humans with congestive heart failure show improvements in 
cardiac function and exercise capacity with GLP-1 administration (82,83). Improvements in 
endothelial function have also been examined in rodents in vivo and ex vivo suggesting a direct 
effect of GLP-1 on endothelial function via eNOS (66-68). In humans, this is manifested in 
consistently lowered blood pressures in some studies (49,50). Thus overall, increasing evidence 
in humans and animals suggests that sitagliptin and other incretin regulatory agents may have 
some cardioprotective effects. Recent observations in rodents and humans suggest that GLP-1 
has a beneficial effect on endothelial and cardiac function (two factors associated with exercise 
function in children and adults with diabetes) (3,48,49,60,63,82,84,87,88). It is plausible therefore, 
that sitagliptin will improve exercise capacity in this group.  
Previous studies in those with diabetes have shown that this exercise impairment is improved, but 
not entirely erased through exercise training.  If improvements in exercise capacity are seen 
resulting from sitagliptin, a greater understanding may be gained of the mechanisms by which 
exercise capacity is reduced in those with diabetes and how they can be reversed. 


Protocol Template 
Page 22 
CF-146, Effective 7/10/11 
 
H. Funding: 
This is an investigator-initiated and protocol with funding and study drug provided by Merck & 
Co., Inc. The protocol was designed, written completely by Drs. Regensteiner and Reusch. The 
protocol will only be performed at University of Colorado. All data collection and statistical 
analysis will be performed by Drs. Regensteiner and Reusch through the University of Colorado. 
Any publications as a result of this research will be submitted by Drs. Regensteiner and Reusch; 
Merck will have no say in what data are published. 
 
I. 
References: 
 
1. 
Abu-Hamdah R, Rabiee A, Meneilly GS, Shannon RP, Andersen DK, and Elahi D. 
Clinical review: The extrapancreatic effects of glucagon-like peptide-1 and related peptides. J Clin 
Endocrinol Metab 94: 1843-1852, 2009. 
2. 
Ban K, Noyan-Ashraf MH, Hoefer J, Bolz SS, Drucker DJ, and Husain M. 
Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through 
both glucagon-like peptide 1 receptor-dependent and -independent pathways. Circulation 117: 2340-
2350, 2008. 
3. 
Basu A, Charkoudian N, Schrage W, Rizza RA, Basu R, and Joyner MJ. Beneficial 
effects of GLP-1 on endothelial function in humans: dampening by glyburide but not by glimepiride. 
AmJ Physiol EndocrinolMetab 293: E1289-E1295, 2007. 
4. 
Bauer TA, Reusch JE, Levi M, and Regensteiner JG. Skeletal muscle deoxygenation after 
the onset of moderate exercise suggests slowed microvascular blood flow kinetics in type 2 diabetes. 
Diabetes Care 30: 2880-2885, 2007. 
5. 
Beckman JA, Creager MA, and Libby P. Diabetes and atherosclerosis: epidemiology, 
pathophysiology, and management. JAMA 287: 2570-2581, 2002. 
6. 
Bell RA, and American Diabetes A. Diabetes 2001 Vital Statistics, American Diabetes 
Association. McGraw Hill Professional Books, 2001. 
7. 
Bhashyam S, Fields AV, Patterson B, Testani JM, Chen L, Shen YT, and Shannon RP. 
Glucagon-like peptide-1 increases myocardial glucose uptake via p38alpha MAP kinase-mediated, 
nitric oxide-dependent mechanisms in conscious dogs with dilated cardiomyopathy. Circulation Heart 
failure 3: 512-521, 2010. 
8. 
Blair SN, Kampert JB, Kohl HW, III, Barlow CE, Macera CA, Paffenbarger RS, Jr., and 
Gibbons LW. Influences of cardiorespiratory fitness and other precursors on cardiovascular disease 
and all-cause mortality in men and women. JAMA 276: 205-210, 1996. 
9. 
Blair SN, Kohl HW, 3rd, Paffenbarger RS, Jr., Clark DG, Cooper KH, and Gibbons LW. 
Physical fitness and all-cause mortality. A prospective study of healthy men and women. JAMA 262: 
2395-2401, 1989. 
10. 
Brandenburg SL, Reusch JE, Bauer TA, Jeffers BW, Hiatt WR, and Regensteiner JG. 
Effects of exercise training on oxygen uptake kinetic responses in women with type 2 diabetes. 
Diabetes Care 22: 1640-1646, 1999. 
11. 
Braun B, Eze P, Stephens BR, Hagobian TA, Sharoff CG, Chipkin SR, and Goldstein 
B. Impact of metformin on peak aerobic capacity. ApplPhysiol NutrMetab 33: 61-67, 2008. 
12. 
Brown MD, Moore GE, Korytkowski MT, McCole SD, and Hagberg JM. Improvement of 
insulin sensitivity by short-term exercise training in hypertensive African American women. 
Hypertension 30: 1549-1553, 1997. 
13. 
Brown SB, Libonati JR, Selak MA, Shannon RP, and Simmons RA. Neonatal exendin-4 
leads to protection from reperfusion injury and reduced rates of oxidative phosphorylation in the adult 
rat heart. Cardiovasc Drugs Ther 24: 197-205, 2010. 
14. 
Celermajer DS, Sorensen KE, Bull C, Robinson J, and Deanfield JE. Endothelium-
dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors 
and their interaction. J AmCollCardiol 24: 1468-1474, 1994. 
15. 
Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd 
JK, and Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at 
risk of atherosclerosis. Lancet 340: 1111-1115, 1992. 


Protocol Template 
Page 23 
CF-146, Effective 7/10/11 
 
16. 
Ceremuzynski L, Chamiec T, and Herbaczynska-Cedro K. Effect of supplemental oral L-
arginine on exercise capacity in patients with stable angina pectoris. AmJ Cardiol 80: 331-333, 1997. 
17. 
Ceriello A, Davidson J, Hanefeld M, Leiter L, Monnier L, Owens D, Tajima N, and 
Tuomilehto J. Postprandial hyperglycaemia and cardiovascular complications of diabetes: an 
update. NutrMetab CardiovascDis 16: 453-456, 2006. 
18. 
Chai W, Dong Z, Wang N, Wang W, Tao L, Cao W, and Liu Z. Glucagon-like peptide 1 
recruits microvasculature and increases glucose use in muscle via a nitric oxide-dependent 
mechanism. Diabetes 61: 888-896, 2012. 
19. 
Committee PAGA. Physical Activity Guidelines Advisory Committee Report, 2008 
Washington, D.C.:  
20. 
Dozier KC, Cureton EL, Kwan RO, Curran B, Sadjadi J, and Victorino GP. Glucagon-
like peptide-1 protects mesenteric endothelium from injury during inflammation. Peptides 30: 1735-
1741, 2009. 
21. 
Emter CA, McCune SA, Sparagna GC, Radin MJ, and Moore RL. Low-intensity exercise 
training delays onset of decompensated heart failure in spontaneously hypertensive heart failure 
rats. American journal of physiology Heart and circulatory physiology 289: H2030-2038, 2005. 
22. 
Fan R, Li X, Gu X, Chan JC, and Xu G. Exendin-4 protects pancreatic beta cells from 
human islet amyloid polypeptide-induced cell damage: potential involvement of AKT and 
mitochondria biogenesis. Diabetes Obes Metab 12: 815-824, 2010. 
23. 
Ferrara CM, Goldberg AP, Ortmeyer HK, and Ryan AS. Effects of aerobic and resistive 
exercise training on glucose disposal and skeletal muscle metabolism in older men. J GerontolA 
BiolSciMedSci 61: 480-487, 2006. 
24. 
Ferreira LF, Lutjemeier BJ, Townsend DK, and Barstow TJ. Dynamics of skeletal muscle 
oxygenation during sequential bouts of moderate exercise. ExpPhysiol 90: 393-401, 2005. 
25. 
Gejl M, Sondergaard HM, Stecher C, Bibby BM, Moller N, Botker HE, Hansen SB, 
Gjedde A, Rungby J, and Brock B. Exenatide alters myocardial glucose transport and uptake 
depending on insulin resistance and increases myocardial blood flow in patients with type 2 diabetes. 
J Clin Endocrinol Metab 97: E1165-1169, 2012. 
26. 
Goyal S, Kumar S, Bijjem KV, and Singh M. Role of glucagon-like peptide- 1 in vascular 
endothelial dysfunction. Indian J Exp Biol 48: 61-69, 2010. 
27. 
Grassi B, Pogliaghi S, Rampichini S, Quaresima V, Ferrari M, Marconi C, and Cerretelli 
P. Muscle oxygenation and pulmonary gas exchange kinetics during cycling exercise on-transitions 
in humans. J ApplPhysiol 95: 149-158, 2003. 
28. 
Green BD, Hand KV, Dougan JE, McDonnell BM, Cassidy RS, and Grieve DJ. GLP-1 
and related peptides cause concentration-dependent relaxation of rat aorta through a pathway 
involving KATP and cAMP. Arch Biochem Biophys 478: 136-142, 2008. 
29. 
Ha JW, Oh JK, Pellikka PA, Ommen SR, Stussy VL, Bailey KR, Seward JB, and Tajik 
AJ. Diastolic stress echocardiography: a novel noninvasive diagnostic test for diastolic dysfunction 
using supine bicycle exercise Doppler echocardiography. JAmSocEchocardiogr 18: 63-68, 2005. 
30. 
Handy JA, Fu PP, Kumar P, Mells JE, Sharma S, Saxena NK, and Anania FA. 
Adiponectin inhibits leptin signalling via multiple mechanisms to exert protective effects against 
hepatic fibrosis. Biochem J 440: 385-395, 2011. 
31. 
Hattori Y, Jojima T, Tomizawa A, Satoh H, Hattori S, Kasai K, and Hayashi T. A 
glucagon-like peptide-1 (GLP-1) analogue, liraglutide, upregulates nitric oxide production and exerts 
anti-inflammatory action in endothelial cells. Diab tologia 53: 2256-2263, 2010. 
32. 
Henry WL, DeMaria A, Gramiak R, King DL, Kisslo JA, Popp RL, Sahn DJ, Schiller NB, 
Tajik A, Teichholz LE, and Weyman AE. Report of the American Society of Echocardiography 
Committee on Nomenclature and Standards in Two-dimensional Echocardiography. Circulation 62: 
212-217, 1980. 
33. 
Hiatt WR, Huang SY, Regensteiner JG, Micco AJ, Ishimoto G, Manco-Johnson M, 
Drose J, and Reeves JT. Venous occlusion plethysmography reduces arterial diameter and flow 
velocity. JApplPhysiol 66: 2239-2244, 1989. 
34. 
Hokanson DE, Sumner DS, and Strandness DE, Jr. An electrically calibrated 
plethysmograph for direct measurement of limb blood flow. IEEE TransBiomedEng 22: 25-29, 1975. 


Protocol Template 
Page 24 
CF-146, Effective 7/10/11 
 
35. 
Home P. Contributions of basal and post-prandial hyperglycaemia to micro- and 
macrovascular complications in people with type 2 diabetes. CurrMedResOpin 21: 989-998, 2005. 
36. 
Huang CY, Shih CM, Tsao NW, Lin YW, Huang PH, Wu SC, Lee AW, Kao YT, Chang 
NC, Nakagami H, Morishita R, Ou KL, Hou WC, Lin CY, Shyu KG, and Lin FY. Dipeptidyl 
peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor 
cells. Br J Pharmacol 167: 1506-1519, 2012. 
37. 
Iwamoto Y, Kuzuya T, Matsuda A, Awata T, Kumakura S, Inooka G, and Shiraishi I. 
Effect of new oral antidiabetic agent CS-045 on glucose tolerance and insulin secretion in patients 
with NIDDM. Diabetes Care 14: 1083-1086, 1991. 
38. 
Jimenez-Feltstrom J, Lundquist I, and Salehi A. Glucose stimulates the expression and 
activities of nitric oxide synthases in incubated rat islets: an effect counteracted by GLP-1 through 
the cyclic AMP/PKA pathway. Cell Tissue Res 319: 221-230, 2005. 
39. 
Jose T, and Inzucchi SE. Cardiovascular effects of the DPP-4 inhibitors. Diabetes & 
vascular disease research : official journal of the International Society of Diabetes and Vascular 
Disease 9: 109-116, 2012. 
40. 
Kasa-Vubu JZ, Lee CC, Rosenthal A, Singer K, and Halter JB. Cardiovascular fitness 
and exercise as determinants of insulin resistance in postpubertal adolescent females. J 
ClinEndocrinolMetab 90: 849-854, 2005. 
41. 
Keller AC, Knaub LA, Miller M, Watson PA, Birdsey N, and Reusch JE. Impact of 
Exercise and Saxagliptin on Mitochondria in the Diabetic Vasculature. Diabetes 2012. 
42. 
Kjaer M, Hollenbeck CB, Frey-Hewitt B, Galbo H, Haskell W, and Reaven GM. 
Glucoregulation and hormonal responses to maximal exercise in non-insulin-dependent diabetes. 
JApplPhysiol 68: 2067-2074, 1990. 
43. 
Knaub LA, McCune S, Chicco AJ, Miller M, Moore RL, Birdsey N, Lloyd MI, Villarreal 
J, Keller AC, Watson PA, and Reusch JE. Impaired response to exercise intervention in the 
vasculature in metabolic syndrome Diabetes and Vascular Disease Research 2012. 
44. 
Kriska AM, Hanley AJ, Harris SB, and Zinman B. Physical activity, physical fitness, and 
insulin and glucose concentrations in an isolated Native Canadian population experiencing rapid 
lifestyle change. Diabetes Care 24: 1787-1792, 2001. 
45. 
Lenski M, Kazakov A, Marx N, Bohm M, and Laufs U. Effects of DPP-4 inhibition on 
cardiac metabolism and function in mice. J Mol Cell Cardiol 51: 906-918, 2011. 
46. 
Liu L, Liu J, Wong WT, Tian XY, Lau CW, Wang YX, Xu G, Pu Y, Zhu Z, Xu A, Lam KS, 
Chen ZY, Ng CF, Yao X, and Huang Y. Dipeptidyl peptidase 4 inhibitor sitagliptin protects 
endothelial function in hypertension through a glucagon-like peptide 1-dependent mechanism. 
Hypertension 60: 833-841, 2012. 
47. 
Loman TG. Advances in body composition assessment. Champaign, Illinois: Human 
Kinetics Publishers, 1992, p. 25-26. 
48. 
Luque MA, Gonzalez N, Marquez L, Acitores A, Redondo A, Morales M, Valverde I, and 
Villanueva-Penacarrillo ML. Glucagon-like peptide-1 (GLP-1) and glucose metabolism in human 
myocytes. J Endocrinol 173: 465-473, 2002. 
49. 
Mafong DD, and Henry RR. Exenatide as a treatment for diabetes and obesity: implications 
for cardiovascular risk reduction. CurrAtherosclerRep 10: 55-60, 2008. 
50. 
Mafong DD, and Henry RR. The role of incretins in cardiovascular control. 
CurrHypertensRep 11: 18-22, 2009. 
51. 
Mannucci E, and Rotella CM. Future perspectives on glucagon-like peptide-1, diabetes and 
cardiovascular risk. Nutr Metab Cardiovasc Dis 18: 639-645, 2008. 
52. 
Maxwell AJ, Schauble E, Bernstein D, and Cooke JP. Limb blood flow during exercise is 
dependent on nitric oxide. Circulation 98: 369-374, 1998. 
53. 
McVeigh GE, Brennan GM, Johnston GD, McDermott BJ, McGrath LT, Henry WR, 
Andrews JW, and Hayes JR. Impaired endothelium-dependent and independent vasodilation in 
patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 35: 771-776, 1992. 
54. 
Monami M, Lamanna C, Desideri CM, and Mannucci E. DPP-4 inhibitors and lipids: 
systematic review and meta-analysis. Adv Ther 29: 14-25, 2012. 
55. 
Monami M, Marchionni N, and Mannucci E. Glucagon-like peptide-1 receptor agonists in 
type 2 diabetes: a meta-analysis of randomized clinical trials. Eur J Endocrinol 160: 909-917, 2009. 


Protocol Template 
Page 25 
CF-146, Effective 7/10/11 
 
56. 
Mosenzon O, and Raz I. Potential cardiovascular effects of dipeptidyl peptidase-4 inhibitors 
in patients with type 2 diabetes: Current evidence and ongoing trials. European Heart Journal, 
Supplement 14: B22-B29, 2012. 
57. 
Murase T, Haramizu S, Ota N, and Hase T. Suppression of the aging-associated decline 
in physical performance by a combination of resveratrol intake and habitual exercise in senescence-
accelerated mice. Biogerontology 2008. 
58. 
Murthy SN, Hilaire RC, Casey DB, Badejo AM, McGee J, McNamara DB, Kadowitz PJ, 
and Fonseca VA. The synthetic GLP-I receptor agonist, exenatide, reduces intimal hyperplasia in 
insulin resistant rats. Diabetes & vascular disease research : official journal of the International 
Society of Diabetes and Vascular Disease 7: 138-144, 2010. 
59. 
Nadeau KJ, Zeitler PS, Bauer TA, Brown MS, Dorosz JL, Drazin B, Reusch JE, and 
Regensteiner JG. Insulin resistance in adolescents with type 2 diabetes is associated with impaired 
exercise capacity. J ClinEndocrinolMetab 2009. 
60. 
Nielsen LL, and Baron AD. Pharmacology of exenatide (synthetic exendin-4) for the 
treatment of type 2 diabetes. CurrOpinInvestigDrugs 4: 401-405, 2003. 
61. 
Nikolaidis LA, Doverspike A, Hentosz T, Zourelias L, Shen YT, Elahi D, and Shannon 
RP. Glucagon-like peptide-1 limits myocardial stunning following brief coronary occlusion and 
reperfusion in conscious canines. JPharmacolExpTher 312: 303-308, 2005. 
62. 
Nikolaidis LA, Elahi D, Hentosz T, Doverspike A, Huerbin R, Zourelias L, Stolarski C, 
Shen YT, and Shannon RP. Recombinant glucagon-like peptide-1 increases myocardial glucose 
uptake and improves left ventricular performance in conscious dogs with pacing-induced dilated 
cardiomyopathy. Circulation 110: 955-961, 2004. 
63. 
Nikolaidis LA, Elahi D, Shen YT, and Shannon RP. Active metabolite of GLP-1 mediates 
myocardial glucose uptake and improves left ventricular performance in conscious dogs with dilated 
cardiomyopathy. AmJ Physiol Heart CircPhysiol 289: H2401-H2408, 2005. 
64. 
Nikolaidis LA, Mankad S, Sokos GG, Miske G, Shah A, Elahi D, and Shannon RP. 
Effects of glucagon-like peptide-1 in patients with acute myocardial infarction and left ventricular 
dysfunction after successful reperfusion. Circulation 109: 962-965, 2004. 
65. 
Nitenberg A, Paycha F, Ledoux S, Sachs R, Attali JR, and Valensi P. Coronary artery 
responses to physiological stimuli are improved by deferoxamine but not by L-arginine in non-insulin-
dependent diabetic patients with angiographically normal coronary arteries and no other risk factors. 
Circulation 97: 736-743, 1998. 
66. 
Nystrom T. The potential beneficial role of glucagon-like peptide-1 in endothelial dysfunction 
and heart failure associated with insulin resistance. HormMetab Res 40: 593-606, 2008. 
67. 
Nystrom T, Gonon AT, Sjoholm A, and Pernow J. Glucagon-like peptide-1 relaxes rat 
conduit arteries via an endothelium-independent mechanism. RegulPept 125: 173-177, 2005. 
68. 
Nystrom T, Gutniak MK, Zhang Q, Zhang F, Holst JJ, Ahren B, and Sjoholm A. Effects 
of glucagon-like peptide-1 on endothelial function in type 2 diabetes patients with stable coronary 
artery disease. AmJ Physiol EndocrinolMetab 287: E1209-E1215, 2004. 
69. 
Ozyazgan S, Kutluata N, Afsar S, Ozdas SB, and Akkan AG. Effect of glucagon-like 
peptide-1(7-36) and exendin-4 on the vascular reactivity in streptozotocin/nicotinamide-induced 
diabetic rats. Pharmacology 74: 119-126, 2005. 
70. 
Park S, Hong SM, and Sung SR. Exendin-4 and exercise promotes beta-cell function and 
mass through IRS2 induction in islets of diabetic rats. Life Sci 82: 503-511, 2008. 
71. 
Parker JC, Lavery KS, Irwin N, Green BD, Greer B, Harriott P, O'Harte FP, Gault VA, 
and Flatt PR. Effects of sub-chronic exposure to naturally occurring N-terminally truncated 
metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 
(GLP-1), GIP(3-42) and GLP-1(9-36)amide, on insulin secretion and glucose homeostasis in ob/ob 
mice. J Endocrinol 191: 93-100, 2006. 
72. 
Poirier P, Bogaty P, Garneau C, Marois L, and Dumesnil JG. Diastolic dysfunction in 
normotensive men with well-controlled type 2 diabetes: importance of maneuvers in 
echocardiographic screening for preclinical diabetic cardiomyopathy. Diabetes Care 24: 5-10, 2001. 
73. 
Quinones MA, Otto CM, Stoddard M, Waggoner A, and Zoghbi WA. Recommendations 
for quantification of Doppler echocardiography: a report from the Doppler Quantification Task Force 


Protocol Template 
Page 26 
CF-146, Effective 7/10/11 
 
of the Nomenclature and Standards Committee of the American Society of Echocardiography. 
JAmSocEchocardiogr 15: 167-184, 2002. 
74. 
Regensteiner JG, Bauer TA, and Reusch JE. Rosiglitazone improves exercise capacity in 
individuals with type 2 diabetes. Diabetes Care 28: 2877-2883, 2005. 
75. 
Regensteiner JG, Bauer TA, Reusch JE, Brandenburg SL, Sippel JM, Vogelsong AM, 
Smith S, Wolfel EE, Eckel RH, and Hiatt WR. Abnormal oxygen uptake kinetic responses in women 
with type II diabetes mellitus. JApplPhysiol 85: 310-317, 1998. 
76. 
Regensteiner JG, Popylisen S, Bauer TA, Lindenfeld J, Gill E, Smith S, Oliver-Pickett 
CK, Reusch JE, and Weil JV. Oral L-arginine and vitamins E and C improve endothelial function in 
women with type 2 diabetes. VascMed 8: 169-175, 2003. 
77. 
Regensteiner JG, Reusch JE, Bauer TA, Quaife RA, Miller TM, Smith SC, and Wolfel 
EE. Recently diagnosed type 2 diabetes mellitus adversely affects cardiac function during exercise. 
MedSciSports Exerc;2009 May;41(5):977-84. 
78. 
Regensteiner JG, Sippel J, McFarling ET, Wolfel EE, and Hiatt WR. Effects of non-
insulin-dependent diabetes on oxygen consumption during treadmill exercise. MedSciSports Exerc 
27: 661-667, 1995. 
79. 
Schneider SH, Amorosa LF, Khachadurian AK, and Ruderman NB. Studies on the 
mechanism of improved glucose control during regular exercise in type 2 (non-insulin-dependent) 
diabetes. Diabetologia 26: 355-360, 1984. 
80. 
Schneider SH, Khachadurian AK, Amorosa LF, Clemow L, and Ruderman NB. Ten-year 
experience with an exercise-based outpatient life-style modification program in the treatment of 
diabetes mellitus. Diabetes Care 15: 1800-1810, 1992. 
81. 
Seibaek M, Vestergaard H, Burchardt H, Sloth C, Torp-Pedersen C, Nielsen SL, 
Hildebrandt P, and Pedersen O. Insulin resistance and maximal oxygen uptake. ClinCardiol 26: 
515-520, 2003. 
82. 
Sokos GG, Bolukoglu H, German J, Hentosz T, Magovern GJ, Jr., Maher TD, Dean DA, 
Bailey SH, Marrone G, Benckart DH, Elahi D, and Shannon RP. Effect of glucagon-like peptide-1 
(GLP-1) on glycemic control and left ventricular function in patients undergoing coronary artery 
bypass grafting. AmJCardiol 100: 824-829, 2007. 
83. 
Sokos GG, Nikolaidis LA, Mankad S, Elahi D, and Shannon RP. Glucagon-like peptide-
1 infusion improves left ventricular ejection fraction and functional status in patients with chronic heart 
failure. J Card Fail 12: 694-699, 2006. 
84. 
Sonne DP, Engstrom T, and Treiman M. Protective effects of GLP-1 analogues exendin-
4 and GLP-1(9-36) amide against ischemia-reperfusion injury in rat heart. RegulPept 146: 243-249, 
2008. 
85. 
Sorensen KE, Celermajer DS, Spiegelhalter DJ, Georgakopoulos D, Robinson J, 
Thomas O, and Deanfield JE. Non-invasive measurement of human endothelium dependent 
arterial responses: accuracy and reproducibility. BrHeart J 74: 247-253, 1995. 
86. 
Tesauro M, Schinzari F, Adamo A, Rovella V, Martini F, Mores N, Barini A, Pitocco D, 
Ghirlanda G, Lauro D, Campia U, and Cardillo C. Effects of GLP-1 on Forearm Vasodilator 
Function and Glucose Disposal During Hyperinsulinemia in the Metabolic Syndrome. Diabetes Care 
2012. 
87. 
Thrainsdottir I, Malmberg K, Olsson A, Gutniak M, and Ryden L. Initial experience with 
GLP-1 treatment on metabolic control and myocardial function in patients with type 2 diabetes 
mellitus and heart failure. DiabVascDisRes 1: 40-43, 2004. 
88. 
Timmers L, Henriques JP, de Kleijn DP, Devries JH, Kemperman H, Steendijk P, 
Verlaan CW, Kerver M, Piek JJ, Doevendans PA, Pasterkamp G, and Hoefer IE. Exenatide 
reduces infarct size and improves cardiac function in a porcine model of ischemia and reperfusion 
injury. J AmCollCardiol 53: 501-510, 2009. 
89. 
Ting HH, Timimi FK, Boles KS, Creager SJ, Ganz P, and Creager MA. Vitamin C 
improves endothelium-dependent vasodilation in patients with non-insulin-dependent diabetes 
mellitus. J ClinInvest 97: 22-28, 1996. 
90. 
Tomas E, and Habener JF. Insulin-like actions of glucagon-like peptide-1: a dual receptor 
hypothesis. Trends Endocrinol Metab 21: 59-67, 2010. 


Protocol Template 
Page 27 
CF-146, Effective 7/10/11 
 
91. 
Vila Petroff MG, Egan JM, Wang X, and Sollott SJ. Glucagon-like peptide-1 increases 
cAMP but fails to augment contraction in adult rat cardiac myocytes. CircRes 89: 445-452, 2001. 
92. 
Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, Bayuga-Ocampo CR, Ham J, Kang 
H, and Evans RM. Regulation of muscle fiber type and running endurance by PPARdelta. PLoSBiol 
2: e294, 2004. 
93. 
Wei M, Gibbons LW, Kampert JB, Nichaman MZ, and Blair SN. Low cardiorespiratory 
fitness and physical inactivity as predictors of mortality in men with type 2 diabetes. AnnInternMed 
132: 605-611, 2000. 
94. 
West A, Green MC, Singel D, Brown M, Newcomer B, Regensteiner J, and Naudeau 
KJ. Exercise Capaity and Muscle Mitochondrial Function in Youth with and without Diabetes. In: 
Pediatric Endocrine Society Annual Meeting. Denver, CO: 2011. 
95. 
Whipp BJ, and Mahler M. Dynaimcs of pulmonary gas exchange during exercise. In: 
Pulmonary Gas Exchange, edited by West JB. New York: Academic Press, 2009. 
96. 
Whipp BJ, and Wasserman K. Oxygen uptake kinetics for various intensities of constant-
load work. J ApplPhysiol 33: 351-356, 1972. 
97. 
Yang L, Li P, Fu S, Calay ES, and Hotamisligil GS. Defective hepatic autophagy in obesity 
promotes ER stress and causes insulin resistance. Cell metabolism 11: 467-478, 2010. 
98. 
Yin JJ, Li YB, Wang Y, Liu GD, Wang J, Zhu XO, and Pan SH. The role of autophagy in 
endoplasmic reticulum stress-induced pancreatic beta cell death. Autophagy 8: 158-164, 2012. 
99. 
Young AA, Gedulin BR, Bhavsar S, Bodkin N, Jodka C, Hansen B, and Denaro M. 
Glucose-lowering and insulin-sensitizing actions of exendin-4: studies in obese diabetic (ob/ob, 
db/db) mice, diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca mulatta). Diabetes 48: 
1026-1034, 1999. 
100. 
Zhao T, Parikh P, Bhashyam S, Bolukoglu H, Poornima I, Shen YT, and Shannon RP. 
Direct effects of glucagon-like peptide-1 on myocardial contractility and glucose uptake in normal 
and postischemic isolated rat hearts. JPharmacolExpTher 317: 1106-1113, 2006. 
101.  
Glueck DH, Muller KE. Adjusting power for a baseline covariate in linear models. Stat. 
Med. 2003;22(16):2535–2551.  
102. 
 Kreidler SM, Muller KE, Grunwald GK, et al. GLIMMPSE: Online Power Computation for 
Linear Models With and Without a Baseline Covariate. Journal of  Statistical Software. 2013; In 
press.