Role of Vascular Function: Oxygen Delivery vs Oxygen Utilization in the Exercise Impairment in Type 2 Diabetes
Phase: NA
Conditions: Type 2 Diabetes
Sponsor: University of Colorado, Denver

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 1 
Effective 10/01/2006 
 
 
Project Title: Role of vascular function: oxygen delivery vs oxygen utilization in the exercise 
impairment in type 2 diabetes  
 
 
 
Principal Investigator:  Judith G. Regensteiner, PhD  
 
Version date:  02/16/2018 
 
 
 
 
 
I. Hypotheses and Specific Aims:  It is well established that functional exercise capacity and 
peak oxygen uptake (VO2) are reduced in patients with type 2 diabetes mellitus (T2DM) compared 
with healthy counterparts (31, 44, 45).  The mechanisms underlying the exercise deficit in T2DM 
remain largely unknown, but previous work has suggested that reduced exercise blood flow (30) 
and impaired submaximal VO2 (44) may be contributing factors.  Both of these findings (30, 44) are 
consistent with a peripheral impairment of skeletal muscle oxygen delivery, oxygen utilization, or 
both.  Indeed, dysfunction of skeletal muscle metabolism plays a key role in the pathophysiology of 
T2DM, and considerable work has described abnormalities of oxidative function in the skeletal 
muscle of people with T2DM (26, 28, 42, 46).  Given this, it is likely that the causes of exercise 
intolerance in T2DM may relate to specific defects at the level of the skeletal muscle, particularly 
given that skeletal muscle blood flow and oxidative capacity are impaired in diabetes.  However, to 
our knowledge, no one has related these peripheral muscle abnormalities to the diminished 
exercise function in this patient group.   
The overarching hypothesis for the proposed research is that both a failure to adequately increase 
muscle oxygen delivery following the onset of exercise and reduced oxidative function of skeletal 
muscle contribute to the acute oxygen deficit and diminished exercise tolerance that has been 
observed in patients with T2DM.  Accordingly, the specific aim of this research proposal is:  
Specific Aim 1. Is the oxygen deficit in diabetes revealed by exercise due to limitation in 
muscle oxygen delivery, muscle utilization or both? 
Rationale. Previous work has demonstrated reduced peak VO2 and slowed VO2 kinetics in people 
with T2DM compared with control subjects (43).  However, the relative contributions of leg blood 
flow versus oxygen utilization limitations could not be evaluated.  Thus, on a systems level, this 
specific aim will evaluate the dynamics of leg blood flow (and thus oxygen delivery) using Doppler 
ultrasound, muscle deoxygenation (O2 extraction) using near infrared spectroscopy (NIRS), and 
pulmonary VO2 during single leg knee extension exercise to identify the predominant mechanisms 
of oxygen delivery versus oxygen utilization abnormalities in the muscle of T2DM during the 
transition from rest to exercise. These studies will generate new information on the determinants of 
muscle oxygen uptake kinetics in diabetes and provide a mechanistic foundation for interpretation 
of improved exercise responses with future interventions such as exercise training.    
Hypothesis 1: Leg blood flow kinetics following the onset of exercise are slowed during exercise in 
people T2DM compared with healthy controls. 
Hypothesis 2: Skeletal muscle deoxygenation (a reflection of oxygen extraction) is more rapid in 
T2DM muscle compared with healthy controls. 
 
 
 
 
 
 
w
COMIRB Protocol 
 
PROTOCOL #: 
06-0062 
(Use Protocol Manager on the COMIRB Website) 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 2 
Effective 10/01/2006 
 
II. Background and Significance: Type 2 
diabetes (T2DM) is the most prevalent 
form of diabetes, affecting nearly 22 million 
people in the United States.  Chronic 
exposure to the dysmetabolic environment 
of diabetes and insulin resistance 
(hyperglycemia, hyperlipidemia, elevated 
oxidative stress, and inflammation) is a 
primary factor in the development of 
diabetic complications.  However, there is 
an unexplained impairment in exercise 
capacity and exercise tolerance in T2DM.  
Despite this gap in our understanding, very 
little research has focused on the potential 
mechanisms underlying exercise 
intolerance in T2DM, particularly as it 
relates to the abnormalities of muscle 
oxidative metabolism and blood flow 
described in T2DM.  This proposal will 
address the mechanisms of exercise 
dysfunction at the level of the peripheral 
skeletal muscle (Figure 1).   
 
Skeletal muscle pathophysiology in 
T2DM.  The pathophysiology of T2DM is 
complex and not completely understood, but early changes in insulin sensitivity of skeletal muscle 
and liver appear to play a major role.  The severity of insulin resistance in T2DM skeletal muscle is 
related to a diminished activity of oxidative enzymes (26), the accumulation of muscle triglycerides 
(27), as well as to reduced electron transport activity of intact mitochondria (28).  There is also 
evidence of capillary basement membrane thickening (55) and a tendency for decreased capillary 
density in T2DM skeletal muscle (24). Whether these structural changes are related to altered 
muscle fiber type composition (i.e. greater proportion of low oxidative type IIb fibers relative to 
highly oxidative type I fibers) (35) and reduced muscle glycogen content in T2DM (19) is unclear.  
However, it appears that both the general ability to deliver oxygen to the skeletal muscle as well as 
the ability of the muscle to utilize oxygen during exercise may be compromised in T2DM. 
Exercise response dynamics.  One method to investigate the potential contributions of muscle 
abnormalities lies in studying the dynamic adaptation 
of the system in response to exercise stress. The 
onset of exercise is associated with an immediate 
demand for energy, which is initially met by pre-
formed high-energy phosphates (creatine phosphate 
and ATP).  However, sustained exercise requires the 
production of ATP via oxidative phosphorylation (and 
hence oxygen consumption)(Figure 2)  (1, 48, 54).  
The time course of increasing aerobic ATP 
production following exercise onset is defined by a 
time constant that is determined by the inherent 
activation, sensitivity, and capacitance of the muscle 
mitochondria (18, 38, 54).  However, once initiated, 
maintaining optimal rates of oxidative 
phosphorylation with continued exercise is dependent on: 1) adequate O2 delivery via enhanced 
exercise blood flow and 2) the ability to increase O2 extraction as described by the Fick principle: 
VO2= Q * (a-vO2 difference). Thus, the time course of increase in muscle VO2 following exercise 
onset (e.g. time constant of VO2 kinetics) is dependent upon both the dynamics blood flow and 
inherent control of mitochondrial respiration. 
Time (seconds)
0
20
40
60
80
100
120
140
160
180
200
0.0
0.2
0.4
0.6
0.8
1.0
free[ADP] 
[PCr] 
muscle VO2 
ATP Turnover
Fractional change 
Figure 2
Figure 1.  The dysmetabolic environment resulting 
from T2Dm is related to endothelial, cardiac, and 
peripheral skeletal muscle dysfunction.  How these 
abnormalities affect exercise capacity and exercise 
tolerance in T2DM remains unclear.
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

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 3 
Effective 10/01/2006 
 
Determinants of VO2 in the exercise transient.  We can assess the systemic integration of 
pulmonary gas exchange and cardiovascular function (e.g. O2 delivery) to meet the O2 utilization 
demands of the exercising muscle through 
measuring the kinetics of pulmonary VO2 during 
the transition from rest to constant work rate 
exercise (3, 44).  In healthy subjects, the time 
constant of pulmonary VO2 kinetics following 
exercise onset exercise is rapid and follows a 
near finite time course, considered to reflect the 
inherent control of mitochondrial oxidative 
metabolism (Figure 3)(12, 14).  However, the VO2 kinetic time constant may become slowed when 
O2 transport and/or O2 utilization of the working skeletal muscle is impaired (e.g. hypoxemia (12, 
25) and cardiopulmonary diseases (41, 51). Thus, in the presence of impaired O2 delivery, 
abnormal muscle metabolic control, or 
reduced muscle oxidative capacity, a slowed 
time constant of VO2 kinetics reflects the 
cumulative effects of these abnormalities. In 
this manner, the measure of VO2 kinetics 
provides an effective measure of the 
integration of O2 supply and utilization with 
exercise, but also describes the functionally 
relevant ability to adjust to the repeated 
demands of submaximal activities 
encountered in daily life. 
Oxygen uptake kinetics in T2DM.  
Previously, we have demonstrated slowed 
pulmonary VO2 kinetic time constants in 
uncomplicated T2DM compared with controls 
(5, 32).  The magnitude of slowed VO2 
kinetics in T2DM was correlated with the 
functional impairment in peak exercise 
VO2, suggesting the mechanisms of 
submaximal and peak exercise limitation 
may be linked. We have also shown that 
exercise training significantly increases 
VO2peak and improves VO2 kinetics (e.g. faster time constant) in women with T2DM, but this does 
not appear to fully normalize the exercise defect in relation to healthy subjects (6).  However, what 
is not known is how peripheral oxygen delivery and/or oxygen utilization of the skeletal muscle may 
influence the exercise response and subsequent limitation in T2DM (Figure 4). A necessary 
approach to address these issues would require measurements be made at the level of the 
exercising muscle.  Previous work in animal models of diabetes have described reduced capillary 
red blood cell flux and a rapid and precipitous fall of microvascular PO2 (mvPO2) in skeletal muscle 
(e.g. muscle oxygenation) following the onset of contractions compared with healthy animals (4, 
29).  Similar observations have recently been made in the skeletal muscle of Goto-Kakazaki rats 
(model of T2DM) (D. Padilla, KSU, personal communication 2006).  These data suggest an initial 
O2 delivery impairment (reflected by the rapid O2 extraction) but also of a significant impairment of 
oxidative metabolism in diabetic skeletal muscle (as later in exercise, MVPO2 rose towards resting 
levels despite continued contractions).  Thus, at least in diabetic animals, there appears to be an 
unexpected dysregulation of O2 delivery to O2 utilization in exercising skeletal muscle.  Similar 
experiments are needed in humans to address these abnormalities in human T2DM.  In this 
proposal, we will utilize an integrated approach of pulmonary VO2, leg blood flow, and measures of 
local muscle oxygenation following the onset of exercise to address the mechanisms of muscle 
oxygen delivery and oxygen utilization in T2DM.  This combination of non-invasive, in vivo 
measurements highlights a novel systems level aspect of the current proposal.    
Healthy Control
Time (s)
-100
0
100
200
300
400
Pulmonary VO2 (ml/min)
0
200
400
600
800
1000
1200
1400
1600
~ Muscle VO2
Figure 3
T2DM Peripheral Muscle 
Dysfunction
Mito capacity & function
Exercise Capacity
Slowed VO2 Kinetics
O2 Utilization
O2 Delivery
?
?
T2DM Peripheral Muscle 
Dysfunction
Mito capacity & function
Exercise Capacity
Slowed VO2 Kinetics
O2 Utilization
O2 Delivery
?
?
Figure 4.  Impairments in muscle oxygen 
delivery secondary to abnormal cardiovascular 
control and/or muscle oxygen utilization due to 
muscle oxidative dysfunction may contribute to 
the observed exercise defect in T2DM.  

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 4 
Effective 10/01/2006 
 
Tools to assess skeletal muscle de-oxygenation kinetics.  Our approach will include a 
relatively new method that offers functional insight into the balance of oxygen delivery to utilization 
of local muscle, near infrared spectroscopy (NIRS).  Similar to measures of mvPO2, changes in 
NIR indices of muscle oxygenation reflect the local balance between O2 delivery and O2 utilization 
as described by the Fick principle where changes in muscle O2 saturation (StO2) and the 
concentration of deoxyhemoglobin/myoglobin ([HHb]) may be considered to reflect changes in 
muscle O2 extraction.  From this principle, the factors that determine VO2 kinetics (e.g. the kinetics 
of blood flow (Q) and muscle O2 extraction (a-v O2 difference)) may be evaluated (11). Modeling 
studies have elucidated the expected profiles of muscle deoxygenation under varying conditions of 
O2 delivery (13).  For example, a primary impairment of muscle O2 delivery relative to O2 utilization 
following the onset of exercise yields predictably faster muscle deoxygenation kinetics (e.g. rapid 
O2 extraction), a result of the poor matching between O2 delivery and O2 demand.  Conversely, an 
inherent impairment of muscle O2 utilization relative to O2 delivery following exercise onset results 
in slowed muscle deoxygenation kinetics (e.g. impaired O2 extraction). Thus, a conceptual 
paradigm for interpretation of muscle deoxygenation kinetics in health and disease has been 
established and can provide directional evidence of the potential contributing mechanisms limiting 
the exercise response.  We will utilize this powerful set of non-invasive measurements to evaluate 
the relative contributions of muscle oxygen delivery and oxygen utilization in relation to the altered 
muscle oxygen uptake responses observed in T2DM. 
Background Summary.  Peak and submaximal exercise responses are impaired in T2DM, the 
mechanisms of which may be related to abnormalities in exercise blood flow (oxygen delivery) 
and/or skeletal muscle oxidative dysfunction (i.e. impaired oxygen utilization).  The relative 
importance of these physiologic system abnormalities in the exercise impairment in T2DM has not 
been resolved.  One factor that may be related to the observed mitochondrial dysfunction and 
potentially the exercise anomaly in T2DM is the reduced skeletal muscle oxidative capacity and 
function.  The purpose of this study is to address the relative importance of these mechanisms and 
establish a background from which the efficacy of interventions such as exercise therapy may 
improve the diabetic condition. 
Innovation.  The proposed study 
will be the first to examine the 
dynamics of oxygen delivery and 
oxygen utilization in T2DM skeletal 
muscle and relate these 
observations to the described 
impairment in T2DM exercise 
performance.  We will be applying a 
new non-invasive technology to this 
important issue in diabetes.  
Further, this study will be the first to 
evaluate parameters of T2DM 
skeletal muscle oxidative function in 
men and women as it relates to 
functional exercise outcome 
parameters. This unique 
combination of techniques has 
strong potential to reveal the 
underlying mechanism disrupting 
exercise capacity in diabetes and 
moves us forward from the 
descriptive studies in this area to 
date. This innovative set of 
experiments will be the beginning of 
an important link between metabolic abnormalities of T2DM and exercise capacity.  
Significance.  It is likely that the causes of exercise intolerance in T2DM may relate to specific 
defects at the level of the skeletal muscle, particularly given that this target organ plays an 
Figure 5. Muscle deoxygenation kinetics in the rest-exercise 
transition in a Healthy control (A) and subject with T2DM (B). 
Exercise begins at time=0. 
Time (sec)
-40
-20
0
20
40
60
80
100
Tissue Oxygen Saturation (StO2, %)
0
20
40
60
80
100
A
baseline
time constant (tau) = 10.4 sec.
Time (sec)
-40
-20
0
20
40
60
80
100
Tissue Oxygen Saturation (StO2, %)
0
20
40
60
80
100
B
baseline
undershoot
tau1 (time constant) = 4.7 sec.
REST
REST
EXERCISE
EXERCISE

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 5 
Effective 10/01/2006 
 
important role in the pathophysiology of insulin resistance and T2DM.  The significance of slowed 
muscle VO2 kinetics following exercise onset relates physical activity encountered in daily life.  As a 
result, slowed VO2 kinetics confer a dependence on non-oxidative energy sources during the early 
portion of exercise causing greater phosphocreatine degradation, a dependence upon non-
oxidative ATP synthesis, and accumulation of hydrogen ion ( [H+]) in skeletal muscle that may, in 
part, contribute to muscle contractile dysfunction, early sensations of muscular fatigue, and a 
reduction in exercise tolerance observed in T2DM patients (49).  It has been suggested that this 
mechanism could predispose persons with T2DM to “feel worse’ when performing exercise, a 
distinct problem given that exercise is a cornerstone of treatment in this population and compliance 
with this treatment is poor (6).  In this context, the characterization of skeletal muscle oxygen 
utilization and hemodynamic responses during exercise will provide meaningful insight into the 
diabetes-specific determinants of functional exercise capacity.  More broadly and from a practical 
standpoint, these data will advance our understanding of the interaction between skeletal muscle 
metabolism and muscle blood flow in the exercise responses in health and T2DM and provide a 
platform upon which to understand skeletal muscle response to different forms of exercise training 
interventions.  
 
III. Preliminary Studies/Progress Report:  
Data supporting hypotheses of Specific Aim1: Muscle deoxygenation kinetics are abnormal 
in T2DM skeletal muscle (Bauer preliminary observations, 2006).  Muscle de-oxygenation kinetics 
were followed during the transition to moderate constant work rate exercise in a sedentary healthy 
control (Figure 5A) and a subject with T2DM (Figure 5B). Both subjects demonstrated similar 
resting muscle oxygen saturation (StO2) prior to exercise onset.  Following the onset of 
contractions and a short time delay, muscle StO2 followed an exponential decline with a time 
constant of 10.4 sec. in the healthy subject and 4.7 sec. in the T2DM subject.  (The T2DM subject 
was associated with slowed VO2 kinetics; e.g. time constant of 55.4 seconds).  The subject with 
T2DM demonstrated an ‘undershoot’ 
of StO2 and subsequent StO2 increase 
towards resting values that was not 
observed in the healthy muscle.  
Interpretation: In the subject with 
T2DM, StO2 decreased more rapidly 
(i.e. more rapid O2 extraction) 
consistent with an initial mismatch of 
muscle O2 delivery to O2 utilization 
(i.e. muscle O2 delivery was slowed).  
In accordance with Fick’s Law of 
diffusion (VO2 = DO2 (PO2capillary – 
PO2Mitochondria)), the rapid fall and 
“undershoot” of muscle StO2 in the 
subject with T2DM could impair 
capillary-myocyte PO2 gradient as 
previously suggested in diabetic 
animals (4).  However, as exercise 
continued, muscle StO2 rebounded 
towards resting levels in T2DM 
muscle (again similar to diabetic 
animals), indicating that later in 
exercise muscle O2 delivery continued 
to increase at a time when O2 
utilization was slowing.  This latter 
response was unexpected given that steady state exercise blood flow to the legs in 
T2DM has previously been shown to be reduced (30); and thus, we would have 
predicted oxygen extraction and muscle deoxygenation to be greater than in control 
(i.e. lower StO2). Indeed, this latter response during the exercise transition could 
Figure 5. Muscle deoxygenation kinetics in the rest-exercise 
transition in a Healthy control (A) and subject with T2DM (B). 
Exercise begins at time=0. 
Time (sec)
-40
-20
0
20
40
60
80
100
Tissue Oxygen Saturation (StO2, %)
0
20
40
60
80
100
A
baseline
time constant (tau) = 10.4 sec.
Time (sec)
-40
-20
0
20
40
60
80
100
Tissue Oxygen Saturation (StO2, %)
0
20
40
60
80
100
B
baseline
undershoot
tau1 (time constant) = 4.7 sec.
REST
REST
EXERCISE
EXERCISE

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 6 
Effective 10/01/2006 
 
reflect an inherent impairment of muscle oxygen utilization due to oxidative 
dysfunction. 
IV. Research Methods 
 
A. Outcome Measure(s):   
Rationale. Previous work has demonstrated reduced peak VO2 and slowed VO2 kinetics in people 
with T2DM compared with control subjects (43).  However, the relative contributions of leg blood 
flow versus oxygen utilization limitations could not be evaluated.  Thus, on a systems level, this 
specific aim will evaluate the dynamics of leg blood flow (and thus oxygen delivery) using Doppler 
ultrasound, muscle deoxygenation (O2 extraction) using near infrared spectroscopy (NIRS), and 
pulmonary VO2 during single leg knee extension exercise to identify the predominant mechanisms 
of oxygen delivery versus oxygen utilization abnormalities in the muscle of T2DM during the 
transition from rest to exercise. 
Experimental plan. The proposed study will determine the leg blood flow kinetics using Doppler 
ultrasound, muscle deoxygenation kinetics using NIRS, and pulmonary VO2 kinetics by metabolic 
cart during plantar flexion exercise. By combining echo measurements with noninvasive estimates 
of pressure, the hydraulic impedance and local stiffness of the aorta (total systemic), brachial (arm) 
and femoral (leg) will be measured using techniques previously developed for pulmonary vessels 
[1-4].  Further, 2-dimesional echo will be used to image the heart, allowing the calculation of 
ventricular strains through speckle tracking methods; strain noninvasively characterizes heart 
function. Insulin sensitivity will be determined using the one stage hyperinsulinemic-euglycemic 
clamp technique (8). Rates of mitochondrial phosphorylation will be assessed by magnetic 
resonance spectroscopy. Muscle biopsies of the gastrocnemeus will be obtained to measure 
muscle histology and mitochondrial respiration. Twenty healthy, overweight sedentary subjects, 15 
lean controls, and 20 subjects with T2DM (specific methods are described below) will be recruited 
for this study.  Mathematical modeling of the measured physiological responses will be used to 
determine the kinetic time constants of each parameter. 
Analysis.  The primary endpoints for Specific Aim 1 are the respective time constants of leg blood 
flow kinetics, vascular impedances and local stiffness, muscle deoxygenation kinetics, and VO2 
kinetics as measured during the transition from rest to constant work rate exercise.  The time 
constant of each parameter, the first three harmonics of each impedance, and elastic modulus for 
each vessel will be used to compare the healthy and T2DM groups using a two sample 
independent t-test with equal variances. Given the small planned sample sizes, should the 
assumption of normality or equal variance of the measures appear unreasonable based on group 
plots, non-parametric tests will be utilized.  To examine the planned correlations between 
measured exercise parameters, correlations (Pearson’s r) and scatter plots will be utilized.  
 
B. Description of Population to be Enrolled:  Study Design and Research Methods   
Recruiting methods. Subjects will be recruited by advertisements approved by COMIRB for this 
purpose. All ads will be approved by COMIRB. Ads will be posted on line, in flyers, in newspaper 
ads, university internet research sites, and radio ads in the Denver area. Appropriate subjects will 
be informed of the research project by the research coordinator and offered to read through the 
consent form prior to study participation.  Process: Individuals will be screened initially by phone 
and all study procedures will be thoroughly explained.  If an individual meets minimum eligibility 
criteria and expresses interest in receiving further information, the consent form will then be 
mailed/delivered for his/her review.  Scheduling for the screening visit will occur after subjects have 
had ample time to review the consent form (at least one week).  When subjects arrive for the 
screening visit, the consent is thoroughly reviewed by the investigator in a quiet setting without 
distractions. Participants are prompted to ask questions and to give their interpretation of the study 
in order to assess their understanding.  Participants will be given a final copy of the signed and 
dated consent form and encouraged to review it often and ask questions as they progress through 
the study.  The Investigators have completed training in the proper methods of obtaining informed 
consent (COMIRB 101). 
Inclusion and Exclusion criteria.  Subjects with T2DM, health lean, and healthy overweight 
sedentary subjects will be enrolled who are between the ages of 30 and 70 years.  Presence of 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 7 
Effective 10/01/2006 
 
T2DM will be confirmed by chart review.  Healthy, sedentary subjects will be defined by normal 
history and physical exam.  Sedentary behavior in all subjects will be objectively defined as not 
participating in a regular exercise program (<one bout of exercise/week) and will be further 
established by use of a questionnaire (Low level Physical Activity Recall, LOPAR).  Subjects will be 
excluded if they have: 1. documented cardiovascular disease in medical history (CAD or PAD), 
ECG findings of cardiac ischemia or conduction system abnormalities with GXT, or other symptoms 
limiting exercise function; 2. uncontrolled hypertension: SBP > 150, DBP> 110. ; 3. Obstructive 
pulmonary disease or asthma, (FeV1 < 0.7);  4. peripheral neuropathy;  5.  Subjects taking beta 
blockers, insulin, or TZD;  6. BMI between 25 and 40 for those with T2DM and overweight controls. 
BMI less than 23 for lean controls;   7. current or past smoking within the last 2 years;  ; 8. Anemia, 
(tHb < 10mg/dl); 9. Autonomic dysfunction (e.g. fall in BP >20mmHg on standing without change in 
HR);   10. Control HbA1c > 5.7; T2DM HbA1c > 10;  11. Type 1 DM; 12. Any implanted metal in 
their body.  
Selection of study population.  Based on local demographic information (Table 1), the expected 
demographics of subjects selected for this study are described in Table 2. 
 
Table 1.  Local demographics in percentages. 
 
American 
Indian or 
Alaskan 
native 
Asian or 
Pacific 
Islander 
Black, not 
of 
Hispanic 
origin 
Hispanic 
White, 
not of 
Hispanic 
origin 
Other or 
unknown 
Total 
Female 
 
 
 
 
 
 
 
Male 
1 
1 
16 
12 
70 
 
100 
Unknown 
 
 
 
 
 
 
 
Total 
1 
1 
16 
12 
70 
 
100 
 
Table 2.  Expected distribution for this study in numbers of subjects. 
 
American 
Indian or 
Alaskan 
native 
Asian or 
Pacific 
Islander 
Black, not 
of 
Hispanic 
origin 
Hispanic 
White, 
not of 
Hispanic 
origin 
Other or 
unknown 
Total 
Female 
 
 
4 
3 
19 
2 
28 
Male 
 
 
4 
3 
19 
1 
27 
Unknown 
 
 
 
 
 
 
 
Total 
 
 
8 
6 
38 
3 
55 
 
Patient accrual.  A total of 55 subjects from the Denver area (15 lean controls, 20 people with 
T2DM, and 20 overweight control subjects) will be enrolled in this study.   
 
Estimated duration of the study.  The estimated duration of participation for each subject in this 
study is 30 hours.  The estimated duration to accrue and evaluate subjects for this study is 2 years. 
 
  
C. Description, Risks and Justification of Procedures and Data Collection Tools: 
 
Treatment, intervention, or observation. This study is designed to observe the exercise 
responses of subjects during moderate intensity exercise. The study will evaluate leg VO2 kinetics 
in patients with T2DM compared with healthy subjects.  Measurements of gases consumed and 
produced, blood flow to the legs at rest and during calf exercise, and muscle tissue saturation by 
non-invasive monitoring will be evaluated.  The use of a similarly-aged control group is to ensure 
that any differences observed between groups were due to the disease of T2DM and not simply a 
result of aging per se. 
Examinations, laboratory tests, procedures, and follow-up visits. 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 8 
Effective 10/01/2006 
 
All patient visits and tests/procedures will take place at the University of Colorado Hospital Clinical 
Translational Research Center. Subjects will come to the GCRC for a total of 8 study visits. 
Visit one:  Screening visit.  Following informed consenting procedures, the visit will include a Low-
level Physical Activity Recall (LoPAR) Questionnaire, a history and physical exam, blood draw and 
urine sample taken for clinical tests, autonomic nervous system testing, dietary survey, and a dual 
x-ray absorptomitrey (DXA).   
Visit two:. Femoral artery flow mediated dilation (FMD), calf reactive hyperemia, femoral artery 
diameter, and calf blood flow at rest and after cuff occlusion will be measured via standardized 
techniques.  Response to sublingual nitroglycerin will serve as a measure of endothelium independent 
vasodilation.   MVC will also be assessed. Cardiac echocardiographic measurements and echo 
measurements. Input impedance and stiffness will be assessed at three systemic vascular 
locations by combining non-invasive measurements of blood flow (PW Doppler) and lumen 
diameter (tissue Doppler) with non-invasive approximations of pressure from arterial tonometry. 
Further, echo images will be taken to obtain right and left ventricular (RV & LV) longitudinal strain, 
LV circumferential strain, and LV torsion. Subjects will perform a graded exercise test (GXT) on a 
stationary bicycle for determination of maximal workrate and VO2max.  Muscle deoxygenation by 
NIRS and arterial oxygen saturation by oximeter, will be monitored at this visit.  Positioning of the 
subjects on the precision isokinetic dynamometer (CSMI, Stoughton, MA), the use of a metronome, 
and practice of plantar flexion at a low resistance will also occur in order to familiarize the subjects 
with the equipment..  
Visit three: A muscle biopsy of the dominant leg gastrocnemius will be performed at this visit. A 
hyperinsulinemic euglycemic clamp, and sphygmocor will be performed in the CTRC during this 
visit . 
Visit four: Five bouts of single leg isotonic plantar flexion exercise will be performed by 
dynamometry on both the dominant and non-dominant leg (total of 10 bouts per subject). Each 
plantar flexion exercise test will be performed for 5 minutes using the dominant leg followed by 6 
minutes of recovery. Resistance will be set as a % of MVC. Two of the four bouts will be at 10% 
MVC, and two bouts will be performed at 20% MVC. Non-invasive measures of common femoral 
artery blood flow by Doppler ultrasound, arterial oxygen saturation by oximeter, and muscle 
deoxygenation by NIRS will be monitored at this visit. Calf blood flow by plethysmography and 
femoral artery FMD will also be assessed. Additionally, one bout of submaximal testing at 20% 
MVC on both the dominant and non-dominant leg will occur while breathing 100% O2. Following 
completion of the isotonic exercises and a 20 minute rest period, three bouts of single leg isometric 
plantar flexion exercises will be performed by the dominant leg only. Contraction during the 
exercise will be at a % of MVC. The first contraction will be for 20% MVC for 120 seconds and the 
second will be for70% MVC for 90 seconds. The third bout will be for 70% MVC for 90 seconds. 
The three bouts will be separated by a rest period. Additionally, two bouts of single leg isometric 
plantar flexion exercise (one at 20% MVC and the other at 70% MVC) will be performed by the 
dominant leg while breathing 100% O2.     
Visit five: During this visit, three bouts of single leg isometric plantar flexion exercise will be 
performed on the dominant leg. Contraction during the exercise will be at a % of MVC. The first 
contraction will be for 70% MVC for 90 seconds, the second will be for 70% MVC for 90 seconds 
and the third will be at 70% MVC for 90 seconds. The three bouts will be separated by a rest 
period. The second bout of single leg isometric plantar flexion exercise (70% MVC) will be 
performed by the dominant leg while breathing 100% O2. The third bout of single leg isometric 
plantar flexion exercise (70% MVC) will be performed 5 minutes after the subject ingests a 
sublingual 300 µg nitroglycerin tablet. During this visit, muscle oxidative function during exercise 
will be assessed via P MRS at rest, during, and in recovery from all plantar flexion exercise tests.  
Exercise Training Period:  Single leg calf muscle endurance training will be performed 5 days per 
week for two consecutive weeks and until visits 6, 7, and 8 are complete.   
Visit six: Femoral artery flow mediated dilation (FMD), calf reactive hyperemia, femoral artery 
diameter, and calf blood flow at rest and after cuff occlusion will be measured via standardized 
techniques.  MVC will also be assessed. Non-invasive measures of common femoral artery blood 
flow by Doppler ultrasound, muscle deoxygenation by NIRS and arterial oxygen saturation by 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 9 
Effective 10/01/2006 
 
oximeter will be monitored at this visit. Calf blood flow by plethysmography and arterial 
stiffness/endothelial function will be non-invasively measured by the Sphygmocor system will also be 
assessed. During this visit, seven bouts of single leg isotonic plantar flexion exercise will be 
performed by dynamometry the dominant and five bouts will be performed on the non-dominant 
leg). On the dominant leg two bouts will be performed at 10% pre exercise training MVC. On both 
the dominant and non-dominant leg two bouts will be performed at 10% post exercise training 
MVC, and two bouts will be performed at 20% post exercise training MVC. Each bout will be 
performed for 5 minutes followed by 6 minutes of rest. Additionally, one bout of single leg isotonic 
plantar flexion exercise will be performed on each leg at 20% post exercise training MVC while 
breathing 100% O2. Following completion of the isotonic exercises and a 20 minute rest period, 
subjects will perform seven bouts of single leg isometric plantar flexion exercises on the dominant 
leg and five bouts of single leg isometric plantar flexion exercises on the non-dominant leg. One 
bout at 20% pre exercise training MVC for 120 seconds will be performed by the dominant leg only. 
One bout at 70% pre exercise training MVC for 90 seconds will be performed by the dominant leg 
only and one bout at 70% post exercise training MVC for 90 seconds will be performed by 
dominant. Each bout will be separated by a rest period. Additionally, a single bout of single leg 
isometric plantar flexion exercise (one at 70% post exercise training MVC) will be performed by the 
dominant leg while breathing 100% O2. 
Visit seven: During this visit, four bouts of single leg isometric plantar flexion exercise will be 
performed on the dominant leg. Contraction during the exercise will be at a % of MVC. The first 
contraction will be for 70% MVC for 90 seconds, the second will be for 70% MVC for 90 seconds 
and the third will be at 70% MVC for 90 seconds. The three bouts will be separated by a rest 
period. The second bout of single leg isometric plantar flexion exercise (70% MVC) will be 
performed by the dominant leg while breathing 100% O2. The third bout of single leg isometric 
plantar flexion exercise (70% MVC) will be performed 5 minutes after the subject ingests a 
sublingual 300 µg nitroglycerin tablet. During this visit, muscle oxidative function during exercise 
will be assessed via P MRS at rest, during, and in recovery from all plantar flexion exercise tests. A 
standardized incremental bicycle exercise test and plethysmography will be performed for the 
determination of systemic exercise capacity. 
Visit eight:  Calf arterial stiffness/endothelial function will be non-invasively measured by the 
Sphygmocor system. A gastrocnemius muscle biopsy will be obtained from untrained and trained calf 
muscles of subjects. 
Optional Visit Nine: Submaximal exercise tests and VO2max test will be performed for O2 uptake 
and heart rate kinetics with NIRS for muscle oxygen extraction. A resting and exercise EKG and vital 
signs will be performed during the visit. 
Optional Visit Ten: Submaximal exercise tests and VO2max test will be performed while breathing 
60% O2 for O2 uptake and heart rate kinetics with NIRS for muscle oxygen extraction. A resting and 
exercise EKG and vital signs will be performed during the visit. 
 
 
Specific Methods. 
Aortic Impedance and Stiffness ultrasound pulse-wave (PW) Doppler images characterizing 
transient blood flow velocity will be obtained in the Aorta from through a parasternal short axis 
approach and in the femoral and brachial vessels using windows described previously. Color M-
mode tissue Doppler images (CMM-TDI) will be used to obtain diameter time-histories of the Aorta 
from the suprasternal notch view and again of the femoral and brachial vessels using standard 
windows.  Both image sets will be obtained using a GE Vivid Seven ultrasound scanner. For the 
PW Doppler images of the Aorta, two dimensional echo and color Doppler will be used to align the 
ultrasound beamline parallel to the main flow direction within the Aorta to minimize errors due to 
ultrasound beam angulation.  In the two peripheral vessels, beamline correction will be utilized due 
to necessity, but will be reduced as much as possible.  The CMM-TDI view of all three vessels will 
provide a long axis representation with the vessel walls perpendicular to the ultrasound beam; 
during acquisition, the beam will be swept through the long axis of the vessel in question to 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
10 
Effective 10/01/2006 
 
determine maximal diameter. Brachial and femoral pressures will be simultaneously measured via 
arterial tonometry (SphygmoCor), while Aortic pressure will be approximated [REF]; each will be 
digitized into the Vivid Seven scanner through its physio port to insure image-pressure synchrony.  
From PW Doppler and pressure, impedance will be obtained, and from CMM-TDI of a vascular wall 
and pressure, stiffness will be obtained; the analysis process for these measurements has been 
well studied and validated [1-5]. 
Arterial Oxygen Saturation.  Oxygen saturation will be assessed in all subjects during all exercise 
tests using an Ohmed oximeter placed on the index finger (Ohmed Corp., Louisville, Colorado). 
Blood collection and preparation:  Up to 4 teaspoons of blood will be drawn for clinical laboratory 
measurements that include markers of insulin resistance (fasting insulin/glucose, c-peptide) and 
standard screening labs (CBC, Cr, lipid panel, urine protein).  Although secondary in nature, the 
requested clinical laboratory markers are necessary to document T2DM disease severity (IR) and 
identify exclusion criteria. 
Biopsy Procedures: Extensive prior work from our lab using muscle biopsies has focused on 
metabolic abnormalities in patients with peripheral arterial disease (47;21).  To measure muscle 
histology and mitochondrial respiration in our patients in the proposed study, we will perform 
skeletal muscle biopsies from the medial aspect of the gastrocnemius as previously described 
(Robbins et al, 2011). A modified Bergstrom needle technique will be utilized to obtain 40-50 
milligrams of skeletal muscle following local anesthesia with 2% lidocaine and a 1cm skin 
incision.(5)  Samples will be embedded in cross-section using optical cutting temperature (OCT) 
tissue freezing medium (Tissue-Tek®, Sakura Finetek USA, Inc., Torrance, CA), snap frozen in 
liquid nitrogen, and stored at -80°C.). Histological analysis will be done using published methods 
(Robbins 2011). The capillary density of each section will be determined by the number of 
endothelial cells/mm2, calculated by dividing the total number of CD31-positive (fluoroscein-
stained) cells by the area (mm2 of tissue) which will be measured with a stage micrometer and 
Image–Pro Plus (Robbins 2011). Dr. Irene Schauer, Investigator on this study, will perform the 
muscle biopsies and also make the mitochondrial respiration measures. Dr. Schauer acquired this 
latter technique at an international OROBOROS workshop in Schroeken, Austria and has a local 
collaborator to assist in optimization of the assay. 
Cardiac Echocardiographic Measurements. Standard two-dimensional and Doppler echocardiography 
will be performed pre and post treatment 85;86 with a commercially available ultrasound system by Dr. 
Jennifer Dorosz, Investigator and Cardiologist on this protocol (GE Vivid 7 Dimension, Milwaukee, 
WI). Chamber sizes, LV end-systolic and diastolic chamber dimensions and wall thickness, fractional 
shortening and left ventricular mass will be quantitated by standard techniques for all individuals. 
Ejection fraction will be calculated using the Method of Disks. In addition to these standard 
measurements, cardiac function will be assessed by tissue Doppler, strain, and strain rate imaging 
using speckle tracking software (Echopak, GE Dimension, Milwaukee, WI). Tissue Doppler of both 
the septal and lateral mitral annuli will measure E' and A' velocities, E':A' ratio, and E:E' ratio. Flow 
propagation will be determined by color M-mode. Global systolic strain (S), systolic strain rate 
(SrS),and diastolic strain rate (SrE and SrA) will be obtained from strain and strain rate curves 
using speckle tracking software19;104;105. 
Consenting procedures. Prior to the screening visit, a copy of the consent form will be mailed to the 
potential subject.  Scheduling for the screening visit will occur after the subject has had the 
opportunity to review the consent form (at least one week).  At the screening visit, informed 
consent will be obtained by the study coordinator/Principal Investigator after a detailed explanation 
of all study procedures/risks and after the subject has had the opportunity to fully review and sign 
the consent form.  The study coordinator/Principal Investigator has completed the COMIRB 101. 
Constant work rate (CWR) exercise. Subjects will perform five-seven bouts of single leg plantar 
flexion exercise performed by dynamometer (CSMI, Stoughton, MA). During visits 4 and 6 each 
plantar flexion exercise test will be performed for 5 minutes using the dominant leg followed by 6 
minutes of recovery. The dynamometer resistance will be set as a % of the individual’s MVC, and 
the frequency of voluntary muscle contractiosn will be 0.5Hz. A rest period will separate each bout. 
During visit 4 two bouts of plantar flexion exercise will be performed at 10% MVC, and three bouts 
will be performed at 20% MVC. All bouts will be performed on both dominant and non-dominant 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
11 
Effective 10/01/2006 
 
legs. During visit 6 two bouts of plantar flexion exercise will be performed at 10% pre exercise 
training MVC, two about at 10% post exercise training MVC, and three bouts will be performed at 
20% post exercise training MVC. The bout at 10% pre exercise training MVC will be performed by 
dominant leg only, all other bouts will be performed by both legs.  
DEXA (Dual-energy X-Ray Absorptiometry): Body composition and bone mineral density will be 
measured using the DEXA technique(53).This technique relies on the absorption of dual electron 
wavelengths for the assessment of body fat, lean tissue, and bone mineral density.  During the 
procedure, the subject will be supine on the measurement table, and the arm of the machine will 
slowly pass over their body.  Body fat distribution will be determined using the waist-to-hip ratio 
where the waist circumference is measured 1/2 the distance from the lowest point of the ribs to the 
iliac crest and the hip circumference is measured at the level of the greater trochanter. 
Dietary interviews:  Customary macronutrient pattern will be ascertained by diet interview on the 
CTRC (94).  A three day diet provided by the CTRC will be used prior to certain visits as stipulated 
in the Visit schedule. The CTRC dietician will prescribe a standardized nutrient breakdown 
including 15% protein, 35% fat and 50% carbohydrate for the three days prior to visits 2, 3, 4, and 
5. The range of sodium in the CTRC diet will be about 1.5 to 1.75 mg/per calorie. In addition, 
sodium intake guidelines will be followed throughout the study to assure standard hydration and 
especially with visits 2, 3, 4, and 5 where the CTRC diet will be utilized for 3 days prior to the visits. 
Endothelial Function: Drs. Regensteiner and Bauer have extensive experience in using FMD to 
assess endothelial function 5;44. FMD will be measured following the protocol of Celermajer 44;106-108 
that measures dilation of the femoral artery by ultrasound in response to hyperemia.  The femoral 
artery diameter will be measured using B-mode ultrasound images, with the use of a GE Vivid 7 
ultrasound system and a 10.0-mHz linear-array transducer as previously reported in our lab (GE 
vivid 7 Dimension, Milwaukee WI.). Scans will be obtained with subjects at rest and following 
reactive hyperemia for assessment of FMD. After measuring responses to cuff occlusion, subjects 
lie quietly for 15 minutes and then sublingual nitroglycerin (300 g) is administered to assess 
endothelium independent vasodilation. 
Equipment Familiarization. Proper positioning of each subject on the precision isokinetic 
dynamometer (CSMI, Stoughton, MA) will be determined. Several single-leg plantar flexion at a low 
resistance along with the use of a metronome will be performed on the precision isokinetic 
dynamometer (CSMI, Stoughton, MA) to familiarize subjects with the equipment and procedure. 
Estimation of microvascular blood flow kinetics.  The estimation of microvascular blood flow 
kinetics will be determined as the time course of Q(capillary), estimated from the rearrangement of 
the Fick equation [Q(capillary) = VO(2)(muscle)/(a-v)O(2)] using the time course of pulmonary 
VO(2) and NIRS derived [HHb] as proxies for VO(2)(muscle) and (a-v)O(2), respectively as 
previously described (14).   
Exercise training. It has been previously shown that muscle oxidative function, enzymes, and 
microvascular structure can be increased following an intensive exercise training protocol 122.  
Following the completion of Visit 5, subjects will undergo supervised single leg, exercise training of 
the index (dominant) calf muscle 5 days per week for two weeks – alternating weight-bearing single 
leg calf raises and single leg calf extensions by endurance resistance training (weight machine 
apparatus).  A prescribed exercise protocol will be completed and supervised by an exercise 
specialist (Dr. Bauer) with close supervision by the Principal Investigator and the study physicians.  
Each session lasts a total of 60 minutes in the Energy Balance Lab at the UC School of Medicine.  A 
14 day time period was chosen because improvements in all exercise performance parameters are 
seen in T2D within this period. For each session, subjects will have a 5 minute warm-up, a 45 minute 
period of moderately intense endurance and resistance exercise (following a prescription set with the 
MVC test conducted before training begins), and a 10 minute cool-down. Subjects will utilize standing 
single leg calf raises repeated to fatigue as well as resistance exercise (single leg calf extensions) 
performed on a weight-stack machine.  Typically, subjects will spend 10-15 minutes on each piece of 
equipment before switching to a different piece of equipment.  Of the 45 minute exercise period, at 
least 30 min of the time will be spent performing the actual single leg exercise training protocol. The 
intensity/duration of each set of calf exercises will be increased on a weekly basis as tolerated, by 
increasing work load and number of single leg calf raises, as applicable. Based on our previous 
experience in this patient population, this program is both tolerable and safe. 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
12 
Effective 10/01/2006 
 
Hyperinsulinemic euglycemic clamp: In order to determine insulin sensitivity, subjects will be 
admitted to the CTRC and fasted for >12 hr during visit 1.  Two intravenous cannulae will be 
established: one for infusions, and the second in retrograde fashion in a dorsal vein of the opposite 
arm for blood sampling. To obtain arterialized venous blood samples, this hand will be kept warm 
with a heating pad. A descending primed continuous infusion of insulin (final 40 mU/m2/min) will be 
initiated at T=0 (about 8AM) and continued until T=180 min for determination of insulin sensitivity. 
Plasma glucose will be maintained at 90 mg/dl by variable infusion of 20% dextrose. An infusion of 
potassium will also be given to maintain normal potassium concentration. The rate of glucose 
infusion will be adjusted based on arterialized blood specimens drawn every 5 minutes, with 
plasma glucose quantified using a ™Analox glucose analyzer at the bedside.  Blood samples will 
also be obtained for determinations of free fatty acids, triglycerides, insulin, and glycerol at 60 min. 
intervals.  After infusion is stopped, the subject will be given a standard meal, and plasma glucose 
levels measured every 15-30 min. for an hour to check for possible hypoglycemia. The subject will 
then be discharged. 
Kinetic Analysis.  Kinetic analysis of pulmonary VO2 and NIRS will be performed as previously 
described (2, 3).  Breath-by-breath VO2, muscle deoxygenation (HHb) will be time averaged to one-
second intervals. Leg blood flow will be evaluated across each contraction.  Mathematical models 
will be employed to fit to the average response curves for VO2, leg blood flow, and muscle O2 
desaturation (StO2 and HHb) using non-linear, least squares regression techniques (Sigmaplot, 
2002) using the following formulas as appropriate for each parameter: X(t)= X(b) + A1 [1- e -(-t-
td/)], X(t)= X(b) + A1 [1- e -(-t-td/1)]+ A2 [1-e-(-t-td2/2)],  X(t)= X(b) + A1 [1- e -(-t-td/1)]+ A2 [1-
e-(-t-td2/2)]+A3 [1-e-(-t-td3/3)] (2, 3, 6, 7, 13) 
Leg Blood Flow.  Leg Blood flow will be determined by pulsed wave Doppler ultrasound in the 
common femoral artery from a site 3-5 cm proximal to the inguinal ligament as described (32, 40, 
50).  Doppler measurements will be carried out using a 5MHz Doppler probe by an experienced 
technician. Serial estimates of mean blood flow will be calculated over each contraction duty cycle 
(0.5Hz) using the mean blood velocity and diameter measurements to enable the determination of 
blood flow dynamics (50).   
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. 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 primarily be used 
in this study as a screen to ensure that all participants are sedentary but will also be used as a 
measure of change in habitual physical activity levels (Attachment). 
Magnetic Resonance Imaging (MRI): For the soleus and tibialis anterior MRS, the subject is 
positioned in the MRI magnet feet first, prone, with the soleus muscle centered in the extremity coil.  
Scout images (usually T1-weighted) position the volume-of interest (usually 2cm x 2cm x 2cm), to 
avoid regions of gross adipose and vascular structures. Spectroscopy acquisition is performed 
using the PRESS pulse sequence without water suppression (TR/TE = 2000/24 ms, 64 averages, 
total acquisition time 2 minutes), and analyzed by SAGE spectroscopy analysis (GE). The water 
peak is fit as a Lorentzian line using Marquardt routine, and subtracted from the spectrum.  The 
remaining peaks (creatinine and choline) are then fit using the Marquardt routine. IMC and EMC 
triglyceride peaks (1.3 and 1.5 ppm, respectively) are obtained from the fit results, corrected for T1 
and T2 relaxation, and expressed as a percentage of the water content(15). Rates of mitochondrial 
phosphorylation will be assessed by 31P magnetic resonance spectroscopy saturation transfer 
performed at 36.31 MHz with the use of a flat, concentric probe made of an inner coil 9 cm in 
diameter (for 31P) and a 13-cm outer coil tuned to proton frequency for scout imaging and shimming 
as previously described(33). Unidirectional rates of ATP synthesis will be measured with the use of 
the saturation-transfer method applied to the exchange between inorganic phosphate and ATP. The 
steady-state magnetization of inorganic phosphate is measured in the presence of a selective 
irradiation of the resonance of ATP and compared with the magnetization of inorganic phosphate 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
13 
Effective 10/01/2006 
 
at equilibrium in a control spectrum (without irradiation of the resonance of ATP)(33).The ratio of 
inorganic phosphate to phosphocreatine in the soleus muscle is also measured by 31P magnetic 
resonance spectroscopy as previously described  (39,52). Subjects will perform two brief (90 
second) muscle contractions against resistance separated by rest while the 31P MRS is performed. 
Dr. Mark Brown, of UCHSC radiology will be responsible for the interpretation of the leg MRS and 
quantitation of IMCL and EMCL as well as mitochondrial measures. Dr. Brown is experienced in 
there procedures and has performed similar interpretations on previous CTRC exercise study of 
adolescents, in subjects with and without diabetes. 
Measurement of Pulmonary Gas Exchange.  Rates of oxygen uptake (VO2) and carbon dioxide 
output will be measured breath-by-breath for the determination of peak VO2 and VCO2 kinetics 
using a Medical Graphics Ultima CPX metabolic system (Medical Graphics Corporation, St. Paul, 
MN).  For all exercise tests, breath-by-breath and data will be collected for three minutes at rest, 
during the entire exercise test, and for a period of seven minutes following exercise.  Heart rate 
(HR) will be recorded simultaneously with ventilatory data via TTL signaling from the ECG recorder.  
Near infrared spectroscopy. Skeletal muscle [HHb] will be assessed by a frequency domain multi-
distance NIRS monitor (Optiplex, Iss, Inc. Chapaign, IL) during each CWR exercise test.  The use 
and limitations of NIRS have been extensively reviewed. 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. The 
NIR data will be sampled continuously at rest and during exercise and recorded at 50Hz.  The 
device probe will be positioned on the lower third of the rectus femoris muscle (approximately 12 
cm above the knee joint) of the dominant limb and secured using a Velcro strap.  The position of 
the probe on the limb will be marked and photographed for identical placement between exercise 
testing visits.  The NIRS monitor is calibrated prior to each visit using a calibration phantom of 
known scattering and optical properties.  Tissue oxygenation data will be collected every 50ms by a 
dedicated data collection computer and saved as text files to a hard disk for later analysis.  This device 
is non-invasive and will be placed only on intact skin. 
Plethysmographic measurements:  Calf blood flow in response to hyperemia will be determined in 
by venous occlusion strain gauge plethysmography (D.E. Hokanson Inc. Issaquah, WA), using 
calibrated mercury-in-silastic strain gauges and expressed as ml/100ml tissue/min as previously 
reported in our laboratory (23,20). Measurements will be made at rest and after reactive 
hyperemia, with the patient supine in bed, in a quiet room at approximately 25oC.  The patient will 
be asked not to eat or drink (NPO) for four hours before the procedure.  The leg will be supported 
at the heart level and a mercury filled silastic strain gauge will be placed on the widest part of the 
calf.  The gauge will be connected to the plethysmograph, calibrated to measure the percent 
change in volume; the plethysmograph in turn will be connected to a chart recording the flow 
measurements in the arm.  For each measurement, a cuff will be placed above the knee and will be 
inflated to approx 35 mm Hg with a rapid cuff inflator (model E-10, Hokanson) to occlude venous 
outflow from the leg.  Every 15 seconds, flow measurements will be recorded for about 7 secs; at 
least 5 separate readings will be taken.   For the reactive hyperemic flows, the leg cuff will be 
inflated to 50 mm Hg above systolic pressure for 5 minutes. 
Pulmonary function test: The metabolic cart (Minneapolis, Minnesota) will be used to perform a pulmonary 
function test. Patient will perform the breathing tests until two FVCs and two FEV1s within 200 ml of each other 
have been obtained.  After a few seconds of normal breathing, following the patient’s breathing pattern as 
displayed, we will instruct the patient to take a big inhale and a big exhale followed a complete inhale and 
exhale.  On the complete exhale, patient should blow out as quickly, forcefully, and completely as possible and 
continue to blow until instructed to stop (at least 6 seconds). This will be repeated as needed. 
Stationary Bicycle Graded Exercise Test. To determine the VO2max and anaerobic threshold, in all 
participants, a graded bicycle protocol to exhaustion will be carried out (44,45).  Each test will have the 
subject seated on the cycle ergometer (Medgraphics, Minneapolis, MN) breathing into the mouthpiece 
of the metabolic cart (Medgraphics, Minneapolis, MN) at rest.  Five minutes of resting data will be 
collected to obtain baseline measurements prior to exercise.  The rest period may be prolonged at the 
discretion of the investigator if additional time is required for adjustment to the mouthpiece and 
stabilization of physiologic variables.  At the start of exercise the work rate will be incremented in 10-25 
watt/min increments and the incremental portion of the test will be 10-14 minutes in duration.  

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
14 
Effective 10/01/2006 
 
Completion of the maximal exercise test on the stationary bicycle in Visit 2 will be utilized to 
determine the maximal workrate, and will use the VO2 data for that time interval to also confirm 
criteria for VO2max.  If criteria for VO2max are not met in the last 15 seconds, we will determine the 
latest 15-second interval where VO2max criteria were met and average the workrate for that 15-
second period as the maximal workrate. VO2max will be defined in the proposed study as VO2 
remaining unchanged or increasing less than 1 ml/kg/min for 30 seconds or more despite an 
increment in work load (53).  If VO2max is not reached by this criterium, it will be repeated on a 
separate day.  The lactate threshold will be estimated from ventilatory data using the V-slope method.  
We will confirm 35% maximal workload is less than the lactate threshold as calculated by the V-slope 
method. One maximal exercise test will be done while breathing 60% O2. 
Submaximal exercise test:  To make VO2 kinetic measurements, constant load bicycling test will 
performed at 85 percent of work-loads below the lactate threshold (steady state).  Each test will begin 
with resting period.  Following this period, the subject will pedal 60 rpm against unloaded 
resistance (e.g. 0 watts) for four minutes immediately prior to beginning the pre-selected work load 
(85% of individual lactate threshold as determined by the V-slope technique) for 5-8 minutes.  This 
will allow all subjects to reach steady-state and echocardiogram measures to be recorded.  The 
work load will be performed up to four times on a given day (with at least 8-30 min rest periods 
between bouts) to enable averaging of VO2 kinetic data within a work load to reduce variability of 
results. These bout will be performed on two separate visits. The first visit will be done while 
breathing regular room air and the second visit will be done while breathing 60% O2.    
Tests of autonomic insufficiency:  To evaluate autonomic insufficiency, we will measure variation in 
RR intervals with cycled breathing as previously reported using accepted methods established in 
our laboratory.  The standard method for obtaining RR variability is as follows: The participant, 
while resting supine, breathes five times per minute, coordinating breaths with a visual electronic 
signal.  This is repeated for five minutes.  To obtain data, maximum inspiratory heart rate (RRi) is 
subtracted from the minimum expiratory heart rate (RRe).  Variations of >30 bpm are considered 
normal, values <20 bpm are considered abnormal.  In addition, autonomic insufficiency will be 
screened measuring lying and standing heart rates and blood pressures (>20 mm fall in upright 
systolic blood pressure without a change in heart rate) as well as immediate heart rate response 
test.  In this test, analysis of the R-R intervals is utilized when going from lying to standing 
expressed as the 30-15 ratio where R-R interval is measured at beats 15 and 30 with a ruler. 
Ventricular Strains: In addition to standard chamber volumes measurements, evaluation of RV & 
LV size and function by strain will be completed by the sonographer.  Off-line speckle tracking 
analysis will be performed by a trained echocardiographer. Speckle tracking of the ventricles will 
focus on the apical four chamber view (LV & RV longitudinal) and the short axis view (LV 
circumferential). Using dedicated software (EchoPak, Vingmed Horton, Norway), the ventricular 
walls will be traced and divided into 6 segments. The tracing will be assessed for adequate 
tracking. In order to obtain valid results, adequate tracking in at least 4 of the 6 segments will be 
verified; otherwise, values from that view will be discarded. Peak strain (S) will be defined as the 
greatest negative deflection on the global strain curve. Peak systolic strain rate (SrS) will be 
defined as the greatest negative deflection on the global strain rate curve. Peak early (SrE) 
diastolic strain rate will be the first positive defection on the strain rate curves. Regional values will 
be an average within each segment. Global values will be obtained from each ventricle as a whole. 
 
Risks 
Insulin Clamp: With the insulin clamp there is the risk of hypoglycemia.  The symptoms of 
hypoglycemia are excessive sweating, faintness, headache, pounding heart, trembling, and 
impaired vision.  Under very extreme degrees of hypoglycemia, a person may lose consciousness.  
Blood glucose levels will be monitored throughout this test to prevent hypoglycemia from occurring. 
I have performed >50 clamps and have caused no episodes of symptomatic hypoglycemia.  
Blood Draws:  Pain may be felt when the needle goes into the vein.  A bruise may form at 
the site. There is a risk of infection. Standard precautions will be used to minimize these risks and 
only CTRC nurses will perform blood draws. 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
15 
Effective 10/01/2006 
 
Fasting: Subjects who are taking a secretagogue run a risk of hypoglycemia during 
fasting. Symptoms listed above will be described in advance and subjects will be instructed not to 
take their AM secretagogue dose on fasting days. 
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. 
IV Risks:  When the needle carrying the plastic tube goes into a vein, it hurts for a short 
time.  The needle is then removed and only the plastic tube remains in the vein.   There will also be 
the minor discomfort of having the plastic tube taped to the arm.  In about one in 10 cases a small 
amount of bleeding under the skin will produce a bruise.  The risk of a blood clot forming in the vein 
is about one in 100, while the risk of infection or significant blood loss is one in 1000. Standard 
precautions will be used to minimize risk and only trained CTRC personnel will place IVs. 
GXT (Graded Exercise Test):  The exercise test is a standard procedure, which we 
routinely perform on hundreds of cardiac subjects every year for diagnosis and evaluation of 
treatment of their condition.  The death rate of this test is approximately one in 10,000 tests and the 
incidence of serious complications including prolonged arrhythmias (irregular heart beats) or 
prolonged chest pain is approximately 4 in 10,000 tests.  This test will be performed on a bicycle. 
The subject may experience slight resistance to breathing due to breathing through mouthpiece. 
Some additional risks of exercising are sore muscles, fatigue, shortness of breath, and 
lightheadedness.  People with diabetes may also experience low blood sugar (hypoglycemia).   
Plethysmography and Femoral Artery FMD: There may be a slight discomfort while the 
arm cuff is inflated. 
Muscle biopsy – A small cut in the skin will be made after using numbing medication on 
the incision site. A small sample will be taken from the calf muscle by pressing a hollow needle into 
the muscle. There is a small chance that subjects could get an infection where the needles goes in.  
There is also a small chance that subjects could have an allergic reaction to the numbing medicine.  
Magnetic Resonance Imaging: 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 can not be tolerated. In addition, any subjects with implanted 
metal can not have an MRI due to the magnet involved. 
Echocardiogram: The risks of the echocardiogram (performed for research purposes) are 
negligible. 
Nitroglycerin: Common side effects of nitroglycerin are bitter taste, headache, 
lightheadedness and palpitations (rapid beating of the heart). 
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. 
Submaximal exercise test: Submaximal exercise test is a standard procedure. The 
participant risk of an abnormal heartbeat or death with this exercise would be similar to the risk 
from walking for 5 minutes at a medium speed, and is far less than the 1 in 10,000 risk for the 
graded exercise test because this test is done at lower work levels.The subject may experience 
slight resistance to breathing due to breathing through mouthpiece. Subjects may also experience 
muscle soreness, fatigue, shortness of breath, chest pain, and lightheadedness during submaximal 
exercise of the calf muscle. Subjects with diabetes may also experience low blood sugar 
(hypoglycemia) while performing submaximal exercise.    
Unforeseen Risks:  This study may include risks that are unknown at this time. 
 
Violation of privacy and loss of confidentiality are both risks to which research participants are 
exposed.  The possibility of these risks increases when protected health information is collected.  
Therefore, using the aforementioned methods, we will strive to maintain patient confidentiality.  No 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
16 
Effective 10/01/2006 
 
sensitive information (domestic violence, child abuse, infectious diseases) will be exposed as part 
of this research. 
 
  
Protected Health Information.  Protected health information collected for this study includes: 
portions of medical records relevant to the study, lab and procedure results (i.e. blood results, 
ultrasound report, exercise test results and the Physical Activity Recall Questionnaire).  Copies of 
all protected health information will be provided to the subject upon request. 
 
    
D. Potential Scientific Problems:   
Pitfalls.  The limitations of NIRS have been extensively detailed in the literature (37). One of the 
major limitations of this method is that, at present, NIRS cannot differentiate between hemoglobin 
in the vascular space and intramuscular myoglobin. In the proposed experiments, we will utilize a 
quantitative tissue oxygen spectrometer (Inspectra, Hutchinson Technologies,  Hutchinson,MN) 
and a phase-modulated, spatially-resolved NIRS spectrometer (Optiplex TS, ISS, Chicago, IL).  We 
are currently validating the non-invasive NIRS methods with invasive hemoglobin saturation and 
blood gas monitoring. Alternatively, we could utilze the invasive methods of rapid areterial-venous 
blood gas sampling and continuous thermodilution across the exercising leg to addres Aim1. 
 
E. Data Analysis Plan:   
Sample Size and Analysis Plan. 
Sample size:  The sample size for this study is based upon our ability to detect a significant 
difference in time constants at 80% power, ά=0.05 and similar variance in the measured time 
constants of leg blood flow and muscle deoxygenation as derived from our previously published 
VO2 kinetics in T2DM and control subjects.  Previous VO2 kinetic data are shown in table 1 (6).  
The time constants for kinetic analysis are 
determined as (1,2,or 3) for the relevant parameter 
analysis (blood flow or muscle deoxygenation) in the 
equations listed above (see Kinetic Analysis).   An 
estimate of the expected blood flow kinetics in T2DM 
and health was calculated from the VO2 and muscle 
deoxygenation responses using the Fick Principle 
(Q= VO2/O2  
extraction) (Table 2.). The pooled variance of the 
time constants for muscle deoxygenation and leg 
blood flow are assumed to be similar to that for VO2 
kinetics (e.g. SD = 5.0s).  With 15 subjects per 
group, we will be able to detect a 5.3sec. difference 
in time constants between groups (at Power =80%, ά 
= 0.05).  Given the predicted time constants for leg blood flow (Table 2) and the expected 
difference between T2DM and control from the muscle deoxygenation preliminary data, our 
planned sample size of 15 subjects per group should be adequate to address the primary 
hypothesis.  Specifically, we will have nearly 100% power to detect the expected difference 
between T2DM and controls for leg blood flow kinetics and 86% power to detect the expected 
difference in muscle deoxygenation kinetics in T2DM and control subjects.  Analysis Plan.  The 
primary endpoints are the respective time constants of leg conduit artery blood flow kinetics and 
muscle deoxygenation kinetics as measured during the transition from rest to constant work rate 
exercise.  The time constant of each parameter will be used to compare the healthy and T2DM 
groups using a two sample independent t-test with equal variances. Given the small planned 
sample sizes, should the assumption of normality or equal variance of the time constants appear 
unreasonable based on group plots, non-parametric tests will be utilized.  The secondary endpoint 
is the time constant of pulmonary VO2 kinetics. To examine the planned correlations between 
measured exercise parameters, correlations (Pearson’s r) and scatter plots will be utilized. These 
exploratory analyses will serve the primary purpose of hypothesis generation for future 
investigations. 
Table 1. VO2 
kinetics 
Study Group 
 
N 
Time constant 
(sec) Mean±SD 
p<0.05 
T2DM 
8 
47.7±4.7* 
Control 
10 
28.8±5.3 
Table 2. BF kinetics 
Study Group 
 
 
Time constant 
(sec)  
Calc Mean  
T2DM 
 
50.0 
Control 
 
27.2 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
17 
Effective 10/01/2006 
 
 
F. Summarize Knowledge to be Gained:   
Expected results and interpretation:  Based upon our previous work, we anticipate VO2 kinetics 
will be slowed during KE exercise in T2DM compared with controls.  Slowed kinetics of leg blood 
flow in T2DM compared with healthy controls would be interpreted as evidence that bulk blood 
flow, and hence oxygen delivery, is impaired following the onset of exercise in T2DM skeletal 
muscle.  As previously discussed in the Background section, if we observe more rapid muscle 
deoxygenation in T2DM muscle, this result would be  consistent with an impairment of local oxygen 
delivery (and blood flow) relative to oxygen utilization.  Conversely, if we observe slowed muscle 
deoxygenation, this result would be consistent with an inherent abnormality in local muscle oxygen 
utilization relative to oxygen delivery in muscle.  Thus, in the proposed experiment, a slowed leg 
blood flow response and more rapid muscle deoxygenation in T2DM muscle would be interpreted 
as support for our working hypothesis (i.e. at least part of the slowed VO2 kinetic response in T2DM 
is attributable to impaired muscle blood flow and oxygen delivery).  
 
G. References: 
 
 
 1.  Barstow TJ, Buchthal S, Zanconato S and Cooper DM. Muscle energetics and pulmonary 
oxygen uptake kinetics during moderate exercise. J Appl Physiol 77: 1742-1749, 1994. 
 2.  Bauer TA, Brass EP and Hiatt WR. Impaired muscle oxygen use at onset of exercise in 
peripheral arterial disease. J Vasc Surg 40: 488-493, 2004. 
 3.  Bauer TA, Regensteiner JG, Brass EP and Hiatt WR. Oxygen uptake kinetics during 
exercise are slowed in patients with peripheral arterial disease. J Appl Physiol 87: 809-816, 
1999. 
 4.  Behnke BJ, Kindig CA, McDonough P, Poole DC and Sexton WL. Dynamics of 
microvascular oxygen pressure during rest-contraction transition in skeletal muscle of 
diabetic rats. Am J Physiol Heart Circ Physiol 283: H926-H932, 2002.    
5.  Bergstrom J. Percutaneous needle biopsy of skeletal muscle in physiological and clinical 
research. Scand J Clin Lab Invest. 1975 Nov;35(7):609-16. 
 6.  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. 
    7. Brozek J, Grande G, Anderson J, Keys A.  Densitometric analysis of body composition: 
revision of some quantitative assumptions.  Ann NY Acad Sci 1963;110:113-140. 
    8. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying 
insulin secretion and resistance. Am J Physiol. Sep 1979;237(3):E214-223. 
 9.  DeLorey DS, Kowalchuk JM and Paterson DH. Relationship between pulmonary O2 
uptake kinetics and muscle deoxygenation during moderate-intensity exercise. J Appl Physiol 
95: 113-120, 2003. 
 
10. DeLorey DS, Kowalchuk JM and Paterson DH. Effect of age on O(2) uptake kinetics and the 
adaptation of muscle deoxygenation at the onset of moderate-intensity cycling exercise. J 
Appl Physiol 97: 165-172, 2004. 
 
11. DeLorey DS, Shaw CN, Shoemaker JK, Kowalchuk JM and Paterson DH. The effect of 
hypoxia on pulmonary O2 uptake, leg blood flow and muscle deoxygenation during single-leg 
knee-extension exercise. Exp Physiol 89: 293-302, 2004. 
 12.  Engelen M, Porszasz J, Riley M, Wasserman K, Maehara K and Barstow TJ. Effects of 
hypoxic hypoxia on O2 uptake and heart rate kinetics during heavy exercise. J Appl Physiol 
81: 2500-2508, 1996. 
 13.  Ferreira LF, Poole DC and Barstow TJ. Muscle blood flow-O(2) uptake interaction and their 
relation to on-exercise dynamics of O(2) exchange. Respir Physiol Neurobiol 147: 91-103, 
2005. 
 14.  Ferreira LF, Townsend DK, Lutjemeier BJ and Barstow TJ. Muscle Capillary Blood Flow 
Kinetics Estimated from Pulmonary O2 Uptake and Near-infrared Spectroscopy. J Appl 
Physiol 2005. 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
18 
Effective 10/01/2006 
 
  15. Fishbein MH, Miner M, Mogren C, Chalekson J. The spectrum of fatty liver in obese 
children and the relationship of serum aminotransferases to severity of steatosis. J Pediatr 
Gastroenterol Nutr 2003;36:54-61. 
 16.  Grassi B, Gladden LB, Samaja M, Stary CM and Hogan MC. Faster adjustment of O2 
delivery does not affect V(O2) on-kinetics in isolated in situ canine muscle. J Appl Physiol 85: 
1394-1403, 1998. 
 17.  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 Appl Physiol 95: 149-158, 2003. 
 18.  Grassi B, Poole DC, Richardson RS, Knight DR, Erickson BK and Wagner PD. Muscle 
O2 uptake kinetics in humans: implications for metabolic control. J Appl Physiol 80: 988-998, 
1996. 
 19.  He J and Kelley DE. Muscle glycogen content in type 2 diabetes mellitus. Am J Physiol 
Endocrinol Metab 287: E1002-E1007, 2004. 
  20. Hiatt WR, Huang SY, Regensteiner JG, Micco AJ, Ishimoto G, Manco-Johnson M. 
Drose J, Reeves JT. Venous occlusion plethysmography reduces arterial diameter and flow 
velocity.  J Appl Physiol 1989;66(5):2239-2244. 
  21. Hiatt WR, Regensteiner JG, Wolfel EE, Carry MR, Brass EP. Effect of exercise training on 
skeletal muscle histology and metabolism in peripheral arterial disease. J Appl Physiol. 1996 
Aug;81(2):780-8. 
  22. Hill JO. Peters JC, Reed GW, Schlundt DG, Sharp T, Greene HL. Nutrient balance in 
humans:  effects of diet composition. Am J Clin Nutr 1991;54:10-17. 
  23. Hokanson DE, Sumner DS, Strandness DE.  An electrically calibrated plethysmograph for 
direct measurement of limb blood flow.  Trans Biomed Eng. 1975;22:25-29. 
 24.  He J, Watkins S and Kelley DE. Skeletal muscle lipid content and oxidative enzyme activity 
in relation to muscle fiber type in type 2 diabetes and obesity. Diabetes 50: 817-823, 2001. 
 25.  Hughson RL and Kowalchuk JM. Kinetics of oxygen uptake for submaximal exercise in 
hyperoxia, normoxia, and hypoxia. Can J Appl Physiol 20: 198-210, 1995. 
 26.  Kelley DE, Goodpaster B, Wing RR and Simoneau JA. Skeletal muscle fatty acid 
metabolism in association with insulin resistance, obesity, and weight loss. Am J Physiol 277: 
E1130-E1141, 1999. 
 27.  Kelley DE and Goodpaster BH. Skeletal muscle triglyceride. An aspect of regional adiposity 
and insulin resistance. Diabetes Care 24: 933-941, 2001. 28.  
Kelley DE, He J, 
Menshikova EV and Ritov VB. Dysfunction of mitochondria in human skeletal muscle in 
type 2 diabetes. Diabetes 51: 2944-2950, 2002. 
 29.  Kindig CA, Sexton WL, Fedde MR and Poole DC. Skeletal muscle microcirculatory 
structure and hemodynamics in diabetes. Respir Physiol 111: 163-175, 1998. 
 30.  Kingwell BA, Formosa M, Muhlmann M, Bradley SJ and McConell GK. Type 2 diabetic 
individuals have impaired leg blood flow responses to exercise: role of endothelium-
dependent vasodilation. Diabetes Care 26: 899-904, 2003. 
 31.  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. J Appl Physiol 68: 2067-2074, 1990. 
 32.  Koga S, Poole DC, Shiojiri T, Kondo N, Fukuba Y, Miura A and Barstow TJ. Comparison 
of oxygen uptake kinetics during knee extension and cycle exercise. Am J Physiol Regul 
Integr Comp Physiol 288: R212-R220, 2005. 
  33.   Lebon V, Dufour S, Petersen KF, et al. Effect of triiodothyronine on mitochondrial energy 
coupling  in human skeletal muscle. J Clin Invest 2001;108:733-7. 
  34. Loman TG.  Advances in Body Composition Assessment.  in Current Issues in Exercise 
Science. Human Kinetics Publishers.  Champaign, Illinois 1992; Monograph 3:25-36. 
 35.  Marin P, Andersson B, Krotkiewski M and Bjorntorp P. Muscle fiber composition and 
capillary density in women and men with NIDDM. Diabetes Care 17: 382-386, 1994. 
  36.   Martin FI HJ. The relationship of acute insulin sensitivity to the progression of vascular 
disease in long-term type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1987;30:149-
53. 

On the Web at http://comirbweb.uchsc.edu 
 
COMIRB Application for Protocol Review (F-???, Revision 8)                                                                                 Page 
19 
Effective 10/01/2006 
 
 37.  McCully KK and Hamaoka T. Near-infrared spectroscopy: what can it tell us about oxygen 
saturation in skeletal muscle? Exerc Sport Sci Rev 28: 123-127, 2000. 
 38.  Meyer RA. A linear model of muscle respiration explains monoexponential phosphocreatine 
changes. Am J Physiol 254: C548-C553, 1988. 
  39.   Meyer RA, Brown TR, Kushmerick MJ. Phosphorus nuclear magnetic resonance of fast- 
and slow-twitch muscle. Am J Physiol 1985;248:C279-87. 
 40.  Moneta GL, Bedford G, Beach K and Strandness DE. Duplex ultrasound assessment of 
venous diameters, peak velocities, and flow patterns. J Vasc Surg 8: 286-291, 1988. 
 41.  Nery LE, Wasserman K, Andrews JD, Huntsman DJ, Hansen JE and Whipp BJ. 
Ventilatory and gas exchange kinetics during exercise in chronic airways obstruction. J Appl 
Physiol 53: 1594-1602, 1982. 
 42.  Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, Miyazaki Y, Kohane I, 
Costello M, Saccone R, Landaker EJ, Goldfine AB, Mun E, DeFronzo R, Finlayson J, 
Kahn CR and Mandarino LJ. Coordinated reduction of genes of oxidative metabolism in 
humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1. Proc Natl 
Acad Sci U S A 100: 8466-8471, 2003. 
 43.  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. J Appl Physiol 85: 310-317, 1998. 
 44.  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. J Appl Physiol 85: 310-317, 1998. 
 45.  Regensteiner JG, Sippel J, McFarling ET, Wolfel EE and Hiatt WR. Effects of non-insulin-
dependent diabetes on oxygen consumption during treadmill exercise. Med Sci Sports Exerc 
27: 661-667, 1995. 
 46.  Ritov VB, Menshikova EV, He J, Ferrell RE, Goodpaster BH and Kelley DE. Deficiency of 
subsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes 54: 8-14, 2005. 
  47. Robbins JL, Jones WS, Duscha BD, Allen JD, Kraus WE, Regensteiner JG, Hiatt WR, 
Annex BH. Relationship between leg muscle capillary density and peak hyperemic blood 
flow with endurance capacity in peripheral artery disease. J Appl Physiol. 2011 Jul;111(1):81-
6. Epub 2011 Apr 21. 
 48.  Rossiter HB, Ward SA, Doyle VL, Howe FA, Griffiths JR and Whipp BJ. Inferences from 
pulmonary O2 uptake with respect to intramuscular [phosphocreatine] kinetics during 
moderate exercise in humans. J Physiol 518 ( Pt 3): 921-932, 1999. 
 49.  Scheuermann-Freestone M, Madsen PL, Manners D, Blamire AM, Buckingham RE, 
Styles P, Radda GK, Neubauer S and Clarke K. Abnormal cardiac and skeletal muscle 
energy metabolism in patients with type 2 diabetes. Circulation 107: 3040-3046, 2003. 
 50.  Shoemaker JK, Hodge L and Hughson RL. Cardiorespiratory kinetics and femoral artery 
blood velocity during dynamic knee extension exercise. J Appl Physiol 77: 2625-2632, 1994. 
 51.  Sietsema KE, Ben-Dov I, Zhang YY, Sullivan C and Wasserman K. Dynamics of oxygen 
uptake for submaximal exercise and recovery in patients with chronic heart failure. Chest 
105: 1693-1700, 1994. 
  52.   Vandenborne K, Walter G, Ploutz-Snyder L, et al. Energy-rich phosphates in slow and fast 
human  
skeletal muscle. Am J Physiol 1995;268:C869-76. 
  53. Weber CT., JS Janicki and PA McElroy.  Cardiopulmonary exercise (CPX) testing.  In: 
Cardiopulmonary Exercise Testing, edited by Weber, CT and Jansiki JS. Philadelphia: WB 
Saunders Company, 1986, p. 151-167. 
 54.  Whipp BJ and Mahler M. Dynamics of Pulmonary Gas Exchange during Exercise. In: 
Pulmonary Gas Exchange, edited by West JB.  New York: Academic Press, Inc., 1980, p. 
33-95. 
 55.  Williamson JR and Kilo C. Capillary basement membranes in diabetes. Diabetes 32 Suppl 
2: 96-100, 1983.