Role of Exercise in the Prevention and Treatment of RAGE-Mediated Inflammation (RECEPTOR) Trial
Phase: NA
Conditions: Diabetes Mellitus, Type 2
Sponsor: University of Michigan

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ROLE OF EXERCISE IN THE PREVENTION AND TREATMENT OF 
RAGE-MEDIATED INFLAMMATION (RECEPTOR) TRIAL 
Principal Investigator: Jacob Haus, PhD 
Draft or Version Number: 1 
Date of Version: 03.20.2019 

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ROLE OF EXERCISE IN THE PREVENTION AND TREATMENT OF  RAGE-
MEDIATED INFLAMMATION (RECEPTOR) TRIAL ___________________________ 1 
BACKGROUND INFORMATION ____________________________________________________ 4 
Rationale ______________________________________________________________________________ 5 
Potential Risks and Benefits _____________________________________________________________ 5 
Study Objectives _______________________________________________________________________ 5 
Study Outcomes _____________________________________________________________________ 6 
Study Hypotheses ______________________________________________________________________ 6 
STUDY DESIGN __________________________________________________________________ 6 
Primary Outcome Measure ______________________________________________________________ 8 
Secondary Outcome Measures ___________________________________________________________ 8 
ELLIGIBILITY AND ENROLLMENT _________________________________________________ 9 
Subject Inclusion Criteria ________________________________________________________________ 9 
Subject Exclusion Criteria________________________________________________________________ 9 
Strategies for Recruitment and Retention __________________________________________________ 9 
Screening Procedures___________________________________________________________________ 9 
STUDY INTERVENTION __________________________________________________________ 10 
Intervention Description ________________________________________________________________ 10 
Group Assignment Procedure ___________________________________________________________ 10 
Aerobic Exercise Training ____________________________________________________________ 11 
Standard Care ______________________________________________________________________ 11 
Reasons for Withdrawal ______________________________________________________________ 12 
Handling of Withdrawals _____________________________________________________________ 12 
Termination of Study ________________________________________________________________ 12 
Accountability Procedures for the Study Intervention _______________________________________ 13 
Concomitant Medications _______________________________________________________________ 13 
Assessment of Subject Compliance with Study Intervention _________________________________ 13 
STUDY SCHEDULE AND PROCEDURES __________________________________________ 14 
Subject Screening _____________________________________________________________________ 14 
Baseline Testing_______________________________________________________________________ 15 
Basal Metabolic Rate ________________________________________________________________ 15 
Maximal Aerobic Capacity ____________________________________________________________ 15 
Metabolic Control Period _____________________________________________________________ 15 
Cognitive Assessment _______________________________________________________________ 16 
Body Composition ___________________________________________________________________ 16 
Oral Glucose Tolerance Test (OGTT) __________________________________________________ 16 
Measurement of Insulin Sensitivity _____________________________________________________ 16 
Urine Collection _____________________________________________________________________ 17 
Skeletal Muscle Biopsy ______________________________________________________________ 17 
Acute Aerobic Exercise Bout: Treadmill Walking _________________________________________ 19 
Microdialysis _______________________________________________________________________ 20 
Follow-up Testing______________________________________________________________________ 21 
Final Study Visit Testing ________________________________________________________________ 21 

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Total Study Tissue Sampling ____________________________________________________________ 22 
Sample Preparation, Storage, and Shipping _______________________________________________ 22 
Assessment of Safety __________________________________________________________________ 23 
Adverse Events _____________________________________________________________________ 23 
Potential Adverse Events and Mitigation Strategies ______________________________________ 23 
Expected Adverse Reactions _________________________________________________________ 25 
Serious Adverse Events and Unanticipated Problems ____________________________________ 25 
Abnormal Laboratory Test Values or Abnormal Clinical Finding Procedure __________________ 25 
Reporting Procedures _______________________________________________________________ 25 
Safety Oversight ____________________________________________________________________ 25 
STATISTICAL CONSIDERATIONS _________________________________________________ 25 
Study Hypotheses _____________________________________________________________________ 25 
Statistics and Sample Size Considerations ________________________________________________ 26 
Safety and Efficacy Review _____________________________________________________________ 26 
Analysis Plan _________________________________________________________________________ 27 
QUALITY CONTROL AND QUALITY ASSURANCE __________________________________ 27 
Ethics and Protection of Human Subjects _________________________________________________ 27 
Informed Consent Process ___________________________________________________________ 27 
Informed Consent/Assent Process (in Case of a Minor) __________________________________ 27 
Subject Confidentiality _______________________________________________________________ 27 
Study Discontinuation ________________________________________________________________ 28 
Future Use of Stored Specimens ________________________________________________________ 28 
DATA HANDLING AND RECORD KEEPING ________________________________________ 28 
Data Management Responsibilities ______________________________________________________ 28 
Data Collection Process ________________________________________________________________ 28 
Study Records Retention _______________________________________________________________ 28 
Protocol Deviations ____________________________________________________________________ 29 
 
 

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BACKGROUND INFORMATION 
Our long-term goal is to identify strategies for the prevention and treatment of 
diabetes. Exercise is a key factor in mitigating diabetic complications. However, the 
molecular foundations of the effects of exercise on diabetes are largely unknown (Figure 
1). Our lab’s specific focus is the receptor for advanced glycation end products (RAGE) 
and its ligand (advanced glycation end products [AGE]). Activation of RAGE, via binding 
of AGEs and other ligands, regulates the development and progression of diabetic 
complications through persistent intercellular signaling (i.e., NF-Kβ) [1]. Targeting RAGE 
directly as a therapeutic strategy has largely been unsuccessful. However, RAGE 
signaling can be interrupted, in vivo, by ADAM10 (a disintegrin and metalloproteinase 10) 
[2, 3], thus creating a soluble isoform of RAGE (sRAGE) that is released from the cell and 
appears into the circulation [3]. Maintaining high levels of circulating sRAGE is 
advantageous as sRAGE sequesters RAGE ligands and prevent cell signaling [4-8]. 
Isoforms of sRAGE (cleaved [cRAGE] and endogenous secretory [esRAGE]), are 
decreased in inflammatory conditions such as type 2 diabetes (T2DM) and coronary 
artery disease, while treatment with recombinant sRAGE, suppresses atherosclerosis 
and vascular dysfunction in animal models of diabetic coronary artery disease [4, 9, 10]. 
Thus, targeting mechanisms that promote sRAGE generation are worth consideration for 
the prevention and treatment of diabetic complications such as coronary artery disease 
and microvascular disease [9, 11].  
 
atRA
Healthy: Physically Active
↑ sRAGE
Vascular Protection
S100s
↑ [Ca2+ ]
Ca2+ sensitive kinase
cRAGE
sRAGE isoforms (decoy receptors)
ADAM10
AGEs
esRAGE
RAGE (membrane bound)
Diseased: Sedentary
↑ RAGE ligands
↓ sRAGE
Vascular Damage
SIRT1
HMGB1
Hyperglycemia
↑ NAD:NADH
ADAM10 mRNA
ADAM10
RARβ
atRA
RXR
atRA
RARβ
RARβ
Ac
RAGE mRNA
NF-Κβ
Transcription of 
inflammatory  cytokines
Nucleus
CRABP 
I/II
Acitretin
Ser PO4 of
ADAM10
Ac
Alternative 
Splicing
Acitretin
atRA
Figure 1 

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Although the exact mechanisms of ADAM10 mediated RAGE release remain 
undefined, calcium related [2, 3] and other signaling (SIRT1) impact ADAM10 [12]. 
Aerobic exercise (AE) presents a unique model for mechanistic study of RAGE release 
as muscle contraction induces robust calcium signaling [13], activates SIRT1 [14-16], and 
provides stimuli for tissue remodeling and resolution of the metabolic profile that drives 
inflammation [17-21]. Our preliminary data show acute AE increases muscle biopsy 
derived ADAM10 activity in lean healthy individuals. We have also shown acute AE 
increases plasma cRAGE in obese, normal glucose tolerant, impaired glucose tolerant, 
and T2DM subjects, while chronic AE (12 weeks) increases skeletal muscle ADAM10 
protein expression, and reduces RAGE protein expression in obese impaired glucose 
tolerant subjects. These findings support the need to explore the effects and molecular 
foundations of AE on AGE-RAGE biology in humans with T2DM. 
 
Rationale 
The rationale for this study was generated using the existing literature base 
coupled with our preliminary data. Capitalizing on the natural ability of skeletal muscle to 
turnover following aerobic exercise, we derived an aerobic exercise model to test our 
central hypothesis that skeletal muscle RAGE release with regular physical activity (via 
ADAM10) is a quantitatively important source of sRAGE appearance into the circulation. 
Once the mechanisms of ADAM10 activation and sRAGE generation are elucidated, 
progress towards the prevention and treatment of diabetic complications may be possible. 
 
Potential Risks and Benefits  
Overall, few potential risks exist. These relate to skeletal muscle biopsies, local 
anesthetic delivery, blood draws, microdialysis of muscle, glucose and insulin infusion, 
strenuous aerobic exercise, body composition assessment (DEXA scan), and data 
confidentiality. Specific details on these potential risks and strategies to mitigate their 
occurrence are discussed in Assessment of Safety and Adverse Events. 
The potential direct benefits include improved health as a result of exercise training 
and potential educational benefits associated with current health status, healthy eating, 
and exercise/physical activity. Each participant may gain knowledge of current health 
status as determined by medical history and physical examination as well as health 
benefits related to monitoring of their diabetes. The benefit to society will include an 
improved understanding of the mechanisms of type 2 diabetes (T2DM) development and 
the treatment of diabetes and reduction of cardiovascular disease risk. Data from this 
study may also help determine an effective approach to prevent the progression of obesity 
and obesity-related diseases and inflammatory conditions. The minimal potential risks are 
outweighed by the benefit of the information that will be acquired from this study. 
 
Study Objectives 
1)  Test the effects of chronic aerobic exercise versus standard care alone on sRAGE in 
the circulation of adults with T2DM. 

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2) Test the effects of chronic aerobic exercise versus standard care alone on RAGE in 
skeletal muscle of adults with T2DM. 
 
Study Outcomes 
The global construct of this study is to provide insight into the role of aerobic 
exercise on muscle health in the context of RAGE biology. Due to the implication of RAGE 
signaling, findings of this study may guide the development of therapeutic modalities for 
T2DM.  
 
Study Hypotheses 
Our central hypothesis is that skeletal muscle contributes to sRAGE appearance 
in the circulation and maintenance of healthy levels of total sRAGE promotes 
cardiometabolic health. Therefore, two specific working hypotheses have been proposed. 
1) Circulating sRAGE levels will increase following 12-weeks of aerobic exercise 
training and this change will be related to reduced muscle RAGE expression and 
increases in insulin sensitivity and cardiorespiratory fitness. 
2) Skeletal muscle expression of RAGE will decrease following 12-weeks of aerobic 
exercise training and this change will be related to increases in circulating sRAGE, 
insulin sensitivity, and cardiorespiratory fitness. 
 
STUDY DESIGN 
Figure 2 provides a general framework of the study design. This 12-week clinical 
study involves the testing and training of middle age to older (40 - 75 y), overweight or 
obese adults (BMI 26 - 44 kg/m2) with type 2 diabetes (T2DM). Sixty subjects will be 
randomized to 12 weeks of aerobic exercise training plus standard care (Aerobic Exercise 
group; n=30) or standard care only (Control group; n=30). Subjects randomized to the 
Control group will be given the option of an off-label exercise intervention. Therefore, 
Control group participants will be allowed to enroll in the AE training group after 
completion of the original Control group trial period. All subjects will undergo the same 
baseline and post testing for basal metabolic rate, maximal aerobic capacity (VO2max), 
body composition, insulin sensitivity, and microdialysis sampling coupled with muscle 
biopsies during an acute aerobic exercise trial before. 
Due to the complexity of this study, an interdisciplinary research team with 
experience in clinical trials, pathophysiology of, and research in obesity, insulin resistance 
and T2DM, vascular function, aerobic exercise testing/training, clinical medicine, 
biotechnology, and advanced biostatistics has been assembled. The research staff has 
many years (>10 y) of direct research experience with human subjects and have been 
trained by experts in the field and University of Michigan protocols and guidelines. 
 

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Type 2 Diabetic Subjects 40-75 y 
(n=60) 
Group Randomization (1:1) 
Aerobic Exercise 
+ 
Standard Care (n=30) 
Standard Care 
 
Control (n=30) 
Baseline Testing: VO2max, body comp, insulin sensitivity 
Acute AE test 
Muscle biopsy 
Microdialysis 
Acute AE test 
Muscle biopsy 
Microdialysis 
Supervised AE training 
1 h/d @85% Hrmax 
Walking, cycling, elliptical, etc 
Free living 
Post Testing: VO2max, body comp, insulin sensitivity 
Acute AE test 
Muscle biopsy 
Microdialysis 
Acute AE test 
Muscle biopsy 
Microdialysis 
Optional off-label  
AE training 
Screening 
Figure 2 

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Primary Outcome Measure 
1) Quantify the change in basal circulating sRAGE after 12-weeks of supervised aerobic 
exercise training. 
Serum will be separated from blood samples collected in vacutainer tubes via 
centrifugation before and after 12-weeks of aerobic exercise training. sRAGE will 
be quantified in these serum samples via ELISA (Quantikine, Human RAGE 
Immunoassay) per manufacture’s protocol. 
 
Secondary Outcome Measures 
1) Quantify the change in basal muscle RAGE expression after 12-weeks of supervised 
aerobic exercise training. 
Basal biopsy derived skeletal muscle RAGE expression will be determined from 
the vastus lateralis before and after 12-weeks of aerobic exercise training. RAGE 
expression will be quantified via Western Blot. Muscle samples (~10 mg) will be 
homogenized and protein concentration will be determined via BCA assay 
(Pierce). Samples (20 μg protein) will be diluted in SDS buffer, heated at 85 °C for 
5 min, resolved via SDS-PAGE (Bio-Rad Laboratories, Hercules, CA) and 
transferred to a nitrocellulose or PVDF membrane. Blocking will occur for 1 h and 
primary antibody (RAGE; Abcam, Cambridge, MA) incubation will occur overnight 
at 4 °C and quantified vs a standard or total protein. 
 
2) Quantify the change in aerobic capacity (VO2max) after 12-weeks of supervised 
aerobic exercise training. 
Maximal oxygen consumption (VO2max) will be established via indirect calorimetry 
during an incremental treadmill test (Cornell protocol) before and after 12-weeks 
of aerobic exercise training. Criteria, such as, rating of perceived exertion >18, 
respiratory exchange ratio >1.10, no further increase in VO2 despite increasing 
workloads, heart rate greater than age-predicted maximum, or volitional fatigue will 
be used to indicate a successful VO2max was achieved. 
 
3) Quantify the change in insulin sensitivity after 12-weeks of supervised aerobic exercise 
training. 
Insulin sensitivity will be established via hyperinsulinemic-euglycemic clamp 
before and after 12-weeks of aerobic exercise training. After the initial basal period 
of this procedure, a primed-continuous infusion (40mU/m2/min) of human insulin 
will be initiated and maintained for a period of 120 min. Glucose levels will be 
clamped at 90 mg/dL by use of a variable glucose infusion (20% dextrose). Blood 
glucose will be measured every 5 min to monitor levels and used to adjust the 
variable glucose infusion rate. The clamp procedure will be completed after the 
120-min period of hyperinsulinemia or until steady state glucose concentration is 
achieved. 

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ELLIGIBILITY AND ENROLLMENT 
A total of 60 adults, (approximately 30 males and 30 females) will complete this 
study. Subjects must be weight stable (±2 kg for ≥6 months), sedentary (<30 min of 
exercise, 3 times per week), and free of any additional contraindications to participation 
in exercise testing or the training. Selection criteria will limit confounding influence on the 
study objectives and exclude subjects with conditions that may have unknown effects on 
metabolic function and the outcomes proposed or the ability to participate in the aerobic 
exercise intervention or testing procedures. Criteria will be reviewed by the research team 
and/or study physician and eligibility to participate will be determined on a case by case 
basis.  
Subject Inclusion Criteria 
• Age 40-75 y  
• Type 2 diabetes 
• Overweight or obese (BMI 26-44 kg/m2)  
• Fluency in English (written and verbal) 
Subject Exclusion Criteria 
• Age <40 or >75 y 
• BMI <26 or >44 kg/m2 
• Existing cardiovascular, cerebrovascular, renal, or hematological disease, cancer, 
or other metabolic diseases that is suspected to impact study outcomes 
• Current use of tobacco  
• Pregnant or lactating 
 
Strategies for Recruitment and Retention 
Subjects will be recruited from the greater Ann Arbor area through multiple 
mechanisms. Primarily, study advertisements will be posted and distributed throughout 
the communities surrounding the University of Michigan. In addition, we will utilize our 
clinical collaborations within the Division of Endocrinology and Metabolism to strengthen 
our ability to identify and recruit potential volunteers. We plan to advertise and seek 
potential volunteers through word of mouth, the Data Office for Clinical and Translational 
Research, MDRC Diabetes Research Registry, the website of the School of Kinesiology, 
newspaper, postal mail, and social media outlets. In our previous studies, we have not 
experienced difficulty recruiting individuals for muscle biopsy or metabolic procedures.  
 
Screening Procedures 
Initial screening of participants will occur via a website screening tool or telephone 
interview. During this time, the study will be described in detail and we will obtain 
information about age, body mass index (BMI), smoking and physical activity status, 
current medical conditions, health status and medications, along with other eligibility 

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criteria. Subjects who meet the initial screening criteria will be scheduled for a complete 
screening at the Michigan Clinical Research Unit (MCRU), Frankel Cardiovascular Center  
(CVC), Domino Farms, or the Central Campus Recreation Building (CCRB). At this time, 
more specific details about the study will be provided along with answers to any questions. 
Each study procedure and all possible risks related to these procedures will be explained 
along with sample exercise programs and explanation of the randomization methods of 
the study. Verbal and written informed consent will be obtained during this initial visit 
according to the guidelines of our Institutional Review Board. A study team member will 
perform a comprehensive medical history. Additionally, participants will complete a 
venipuncture blood draw and urine collection for laboratory assessment of complete blood 
count (CBCD), comprehensive metabolic panel (COMP), urinary analysis (UA), urinary 
microalbumin (UMA), hemoglobin A1C (A1C), thyroid stimulating hormone (TSH), and 
lipid panel. Females will also be asked to provide urine for a pregnancy test (for pre-
menopausal women). In addition, participants will complete a supervised 12-lead resting 
electrocardiogram and graded exercise stress test (with VO2) at the CVC or Domino 
Farms. EKG test results will be reviewed by a study physician. If any blood work or EKG 
results are clinically significant, the participant will be notified and referred to their 
personal physician for additional evaluation prior to further consideration for participation 
in this study. Baseline food intake will be assessed from diet history including multiple 
replicate 24-hour recalls to estimate usual intake, customary eating pattern, and food 
frequency questionnaires. After confirmation of the participant meeting all inclusion and 
exclusion criteria, participants will be enrolled into the study intervention. 
 
STUDY INTERVENTION 
Intervention Description 
This study’s intervention includes 12 weeks (1 week) of supervised aerobic 
exercise training for the group randomized to Aerobic Exercise. The Control group will not 
be subject to an intervention as they will receive standard care only (see Standard Care). 
Subjects randomized to the Control group will be given the option of an off-label exercise 
intervention. Therefore, Control group participants will be allowed to enroll in the AE 
training group after completion of the original Control group trial period. 
 
Group Assignment Procedure 
Following enrollment into the study, participants will be randomized into either the 
Aerobic Exercise group (n=30) or Control group (n=30. Females and males will be equally 
distributed between the groups. Regardless of group assignment, each participant will 
receive standard care for diabetes management. 
 
 
 

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Aerobic Exercise Training 
Every AE training session will be supervised by the research team using 
established protocols [22-28]. Training sessions will take place at the Central Campus 
Recreation Building (CCRB), Domino Farms, or other University of Michigan recreation 
facility (e.g., North Campus Recreation Building) approximately 5 days per week and 
include treadmill walking, cycling, elliptical, or other related modes of exercise. Briefly, AE 
training will begin with a 2-week ramp-up, familiarization period of progressing exercise 
intensity and duration. Training intensity will progress from 55% VO2max for week 1 (40 
min session), to 60-65% VO2max for week 2 (50 min session), to ~70% VO2max for all 
other weeks (50 min session). Subjects will perform a brief warm-up and cool-down (~5 
min each) that may include stretching or other light exercises for a total exercise time of 
60 min. Subjects will wear heart rate monitors during each training session to provide 
real-time feedback of target heart rate. Intensity, duration, resting and exercise heart 
rates, and blood pressures will be recorded for each session. Exercise duration and 
intensity may be adjusted at the discretion of the PI, Co-I, or study team member. Follow-
up VO2max tests will be performed at approximately weeks 4 and 8 to monitor progress. 
AE intensity will be adjusted to match the prescribed training intensity [23, 24, 29-32]. 
Subjects in the AE training group will also conduct foot safety checks with the research 
team to monitor foot ulcers/blisters. Relevant findings during foot inspections will be 
addressed with the participant and study physician. Non-weight bearing exercise may be 
implemented if the subject is limited. 
As a contingency, exercise training may be done remotely. At the PI, research 
team member, or study physician’s discretion, participants may conduct exercise training 
at their residence, local gym, or other appropriate facility. During these training sessions, 
a study team member will communicate with the participant prior to the session and 
discuss the goals and training plan. More specifically, a team member will discuss where 
the session is to occur, the duration of the session, mode of exercise, and exercise 
intensity. During the session, a team member may monitor exercise via telephone or 
video chat (e.g., zoom). Exercise intensity is monitored via a HR monitor. Each participant 
wears a Polar HR monitor watch for the duration of the study and is familiarized with its 
use. If a remote session is to occur, the participant will be sent home with a  heart rate 
strap that communicates with the watch to give HR feedback (i.e., via mail, during visit to 
the lab, or meeting a local establishment, etc). HR is digitally recorded and downloaded 
as described elsewhere (another form of monitoring exercise).   
 
Standard Care 
All subjects will receive standard care for diabetes management as directed by 
their individual physician and diabetes health care team according to American Diabetes 
Association, Standards of Medical Care in Diabetes position statement, 2018 [33]. All 
newly diagnosed subjects with type 2 diabetes will be encouraged to receive Diabetes 
Self-Management Education. In addition, both groups will be given a wrist worn device 
during the intervention and trained in their use to track physical activity patterns. Subjects 
will meet approximately twice monthly with the study team to download accelerometer 
data. 

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Reasons for Withdrawal 
Participants are free to withdraw their consent and discontinue participation at any 
time. We do not anticipate participant withdrawal due to study-related reasons as similar 
exercise training interventions have been taking place for decades with high levels of 
adherence. Additionally, we have extensive experience managing clinical trials in the 
proposed population [26, 30, 34, 35]. However, we recognize participants interests, 
health, and schedules may change. The research team’s best effort will be made to 
accommodate the participants needs and minimize attrition. The following are potential 
reasons for withdrawal: 
• Time Commitment: Some participants may find the time commitment associated 
with this study too burdensome for their schedule. The study coordinator will 
discuss this time commitment prior to enrollment.   
• Schedule Conflicts: Some participants may find it difficult to determine times they 
are available to come in for study visits. The study coordinator will also discuss 
preferred visit start times prior to enrollment to limit potential schedule conflicts.  
• Discomfort: Participants may experience discomfort during blood and tissue 
sampling procedures and wish to discontinue the study. Prior to enrollment, the 
study coordinator will discuss all procedures and answer any questions 
participants may have concerning sampling procedures.  
• Unforeseen Reasons: There may be other reasons participants may withdrawal 
from the research study that are unknown at this time.  
 
Handling of Withdrawals 
The participants right to withdraw from the study will be respected and will occur 
within the timeframe they request. In the event of a participant withdrawal, samples and 
data collected during their participation will be stored for future use or destroyed, based 
on the participants request. 
 
Termination of Study  
The research staff reserves the right to suspend/terminate subject’s participation 
in this study at any time. This decision will be communicated to the participant and a 
reasonable effort will be made to mitigate/avoid this outcome. Potential study 
suspension/termination reasons include: 
• Ineligibility: The subject’s eligibility changes during the study 
• Safety: The research has become harmful to the participant 
• Health: The subject’s health condition changes and requires treatment which 
affects the study outcomes 
• Incompliance: Subject fails to follow the study guidelines 
• Cancelation: The research study is canceled or suspended 
 
 

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Accountability Procedures for the Study Intervention 
The research team will communicate internally weekly to discuss study progress 
and ensure all objectives and guidelines are met. From a participant perspective, 
agreement to enroll in the study will necessitate adherence to all study guidelines. The 
research staff will have regular contact (approximately 5 days/week) with subjects in the 
Aerobic Exercise group and will meet approximately twice monthly with the control group 
to monitor habits and study progress. We have found these interactions are indispensable 
for study adherence. 
 
Concomitant Medications 
Subjects participating in this study will be diagnosed with T2DM and therefore have 
unique medical needs. In addition to the screening provided in this study, each participant 
will be encouraged follow their personal physician’s guidance. Prior to and during the 
intervention period, all medication use, dose and frequency will be documented 
 
Assessment of Subject Compliance with Study Intervention 
Subject compliance will be regularly assessed and documented by the research 
team. Participants enrolled in the AE group will have contact with the research team 
approximately 5 days per week and the Control group will have contact with the research 
team approximately twice per month. Compliance will be determined through routine 
personal communication and primarily evaluated based on exercise training attendance 
(Aerobic Exercise group only), physical activity tracking, and dietary monitoring. Subjects 
in the Aerobic Exercise group are expected to complete >80% of the training sessions 
over the course of the 12 weeks. 
 

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STUDY SCHEDULE AND PROCEDURES 
Figure 3 provides a brief study summary of the study schedule. This study is 
composed of initial subject screening and enrollment based on established inclusion and 
exclusion criteria and randomization to 12 weeks of Aerobic Exercise training or Control 
Period (both including established Standard Care practices). Participants will all complete 
the same baseline testing and final study visit testing.  Subjects randomized to the AE 
group will also undergo follow-up testing at weeks 4 and 8 (1 week) to adjust exercise 
training parameters. All testing will occur at the Michigan Clinical Research Unit (MCRU), 
Michigan Nutrition Obesity Research Center (NORC), Domino Farms, Frankel 
Cardiovascular Center (CVC), and/or Central Campus Recreation Building (or other 
University recreation building) and based on subject schedules, will include a total of 
approximately 7 visits. 
 
Scheduling Flexibility 
Due to limitations of the subject’s schedule, study team, experimental approach, 
and/or clinic availability, some procedures may be excluded or adjusted from any given 
visit. Excluded or adjusted procedures will be at the discretion of the PI or Co-I and will 
not be considered a protocol deviation. 
 
Subject Screening 
Initial screening of participants will occur via a website screening tool or telephone 
interview. During this time, the study will be described in detail and we will obtain 
information about age, body mass index (BMI), smoking and physical activity status, 
current medical conditions, health status and medications. Subjects who meet the initial 
screening criteria will be scheduled for a complete screening at the MCRU, CVC, Domino 
Farms, and/or CCRB. Verbal and written informed consent will be obtained during this 
Figure 3 

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15 
initial visit according to the guidelines of our Institutional Review Board. Subjects will be 
screened based on the inclusion and exclusion criteria. Screening procedures include a 
comprehensive medical history examination, blood draw, resting electrocardiogram, 
graded exercise stress test, and urine collection. In addition, pre-menopausal women will 
have a urine pregnancy test. All screening findings will be reviewed by the research team 
and study physician(s) (or CVC/Domino Farms physician) when appropriate. 
 
Baseline Testing 
Baseline testing procedures will be supervised by the research team and 
completed at the MCRU, CVC, Domino Farms, CCRB, and/or NORC. Testing includes 
measurement of basal metabolic rate, maximal aerobic capacity, cognitive assessment 
questionnaire (NIH ToolBox), body composition, OGTT, and insulin sensitivity. In addition, 
the Minnesota Leisure Time Physical Activity and Seven Day Physical Activity Recall 
Questionnaires will be administered to assess habitual physical activity levels. Subjects 
will also undergo an acute aerobic exercise bout (treadmill walking) coupled with 
microdialysis and muscle biopsies. Each of these testing procedures are critical for 
completion of the study objectives and evaluation of the study hypotheses. 
 
Basal Metabolic Rate 
Basal metabolic rate will be measured at Domino Farms, MCRU, CCRB, or CVC 
by the research team and/or medical staff. Subjects will initially rest quietly in their room 
for ~30 min. We will then measure their basal metabolic rate using a canopy-equipped 
metabolic cart for ~20 min.    
 
Maximal Aerobic Capacity 
Maximal oxygen consumption (VO2max) will be captured during the GXT and will 
be used as the criterion measure of aerobic capacity (i.e., physical fitness). The VO2max 
test will be performed using a Cornell (or similar) protocol at Domino Farms or the CVC. 
Measurements of VO2max and maximal heart rate (HRmax) will be performed at baseline 
and following the 12-week intervention period (interim visit, approximately one week 
before the final study visit). In the Aerobic Exercise group, baseline VO2max will used to 
determine the starting training intensity and VO2max tests will be repeated at 
approximately 4 and 8 weeks of aerobic exercise training to adjust target HR. The control 
group will also return at approximately weeks 4 and 8 as well, as a blood draw (~20 ml) 
will also be taken for all individuals at these time intervals.  
 
Metabolic Control Period 
The metabolically sensitive pre- and post-intervention insulin sensitivity 
assessment (glucose clamp) will be performed during a 1-night inpatient stay at MCRU 
(two 1-night stays total over the course of the study). The goal of this overnight stay is to 
control metabolism to increase outcome validity of the insulin sensitivity assessment. 

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Participants will be asked to reside in the MCRU for 1 night and will receive a weight-
maintenance isocaloric diet (55% carbohydrate, 30% fat, and 15% protein) provided by 
the study team or MCRU/NORC metabolic kitchen. Subjects will be asked to refrain from 
physical activity outside of their normal activities of daily living during this control period 
Additionally, subjects will complete an OGTT, questionnaires, body composition (DEXA 
scan), and acute exercise test within ~1 week of this overnight visit at the CVC, MCRU, 
CCRB, and/or Domino Farms. Metabolic measures will be performed >24 h after the last 
exercise bout. This approach has been successfully used by the research team in human 
metabolic studies to control for effects of diet and physical activity [23-28, 31, 35-37]. 
 
Cognitive Assessment 
 
Participants will complete an NIH developed cognitive assessment tool (ToolBox) 
to evaluate cognition on an iPad. This assessment will be administered by the research 
team. Dr. Brian Callaghan MD (Neurology) will oversee administration and interpretation 
of this assessment tool.  
 
Body Composition  
Body composition will be assessed by dual-emission X-ray absorptiometry 
(DEXA). DEXA scans determine components of body composition such as bone mineral 
density, lean body mass, fat mass, and segmental adiposity such as truncal fat and 
visceral fat mass. DEXA scans will be performed in the MCRU by the MCRU staff or a 
member of the study team. 
 
Oral Glucose Tolerance Test (OGTT) 
 
This standard test is utilized to detect and confirm the presence of diabetes. Briefly, 
a polyethylene catheter will be inserted into an antecubital or dorsal hand vein and the 
subject will drink a beverage containing 75 g of glucose. If a catheter is not successfully 
inserted, venipuncture for blood collection may be implemented. In addition, finger-sticks 
may also be utilized to assess blood glucose levels. A baseline blood draw will precede 
the consumption of the glucose drink along with blood draws approximately every 30 min 
for 3 h. Current use of physician confirmed diabetes medication, fasting glucose values ≥ 
126 mg/dL and 2 hr OGTT values ≥ 200 mg/dL will be used as the criteria for type 2 
diabetes [38]. Blood samples will be collected and immediately transferred to vacutainers, 
centrifuged, and stored at -80°C until analysis for blood glucose and insulin 
concentrations. OGTTs will take place at MCRU, CCRB, or Domino Farms and will be 
supervised by members of the study team after catheter insertion by the clinical staff or 
qualified research team member. 
 
Measurement of Insulin Sensitivity  
Whole body insulin sensitivity will be assessed using a 40 mU/m2/min 
hyperinsulinemic-euglycemic (glucose 90 mg/dL) clamp procedure as described 

 _____________________________________________________________________________________________ 
17 
previously and used routinely by the research team [23-27, 31, 34, 35]. Briefly, a 
polyethylene catheter will be inserted into an antecubital vein for infusion of insulin and 
glucose. A second catheter will be inserted retrograde into a dorsal hand vein with the 
hand warmed in a heated box for blood sampling [39]. Once catheters have been inserted, 
basal blood draws will take place. After the basal period, a primed-continuous infusion 
(40 mU/m2/min) of human insulin will be initiated and maintained for a period of 
approximately 120 min. Glucose levels will be clamped at approximately 90 mg/dL by use 
of a variable glucose infusion (20% dextrose). Blood glucose will be measured 
approximately every 5 min to monitor levels and used to adjust the variable glucose 
infusion rate. The clamp procedure will be completed after once steady state glucose 
concentration is achieved. Upon completion, insulin infusion will be discontinued while 
glucose infusion is continued until plasma glucose stabilizes. A comprehensive metabolic 
panel (COMP) will be assessed before and after the clamp procedure. In addition, the 
subject will be provided with a post-clamp meal or snack that consists of carbohydrate 
and protein to further maintain blood glucose (e.g., juice, sandwich). This procedure will 
take place at MCRU. 
 
Urine Collection 
Subjects will be asked to collect their urine starting at approximately 4:00am on 
the morning of the  hyperinsulinemic-euglycemic procedure. Urine samples will be 
collected in standard urine collection vessel. Total volume and time of collection will be 
recorded. 
 
Skeletal Muscle Biopsy 
The modified muscle biopsy technique was introduced nearly 60 years ago [40] 
and is a fundamental component of muscle health research (e.g., insulin sensitivity, 
inflammation). This technique is used commonly in the medical research field (as 
indicated by large scale consortiums [e.g., NIH funded MoTrPAC]) and is currently utilized 
at the University of Michigan. Members of the research team have been involved with 
muscle biopsies for ~15 y, totaling >500 muscle biopsies in populations including athletes, 
elderly, obese, type 2 diabetics [23, 35, 41-46]. Dr. Jacob Haus, PhD and Dr. Jeffrey 
Horowitz, PhD will perform the muscle biopsies which are critical to completion of this 
study’s objectives. 
Muscle biopsies will be obtained before and within 30 minutes after the acute 
exercise bout. These exercise trials will take place before and after 12 weeks of the 
aerobic exercise intervention or standard care periods (4 total muscle biopsies per 
subject). Muscle samples (~200 mg; approximately the size of a pea) will be obtained 
from the vastus lateralis muscle (thigh muscle) following local anesthetic delivery using 
the modified muscle biopsy technique [40, 41, 44, 47]. Muscle tissue will be quickly 
trimmed of excess connective tissue and fat, blotted with gauze to remove blood and 
processed specific to future analysis. Subjects will be instructed on how to care for the 
wound and supplied with ice and pressure bandages after each biopsy to reduce 

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18 
inflammation and any soreness. A member of the research team will follow-up with each 
participant the day after a muscle biopsy. 
 
SOP for skeletal muscle biopsy 
• Anti-coagulant medication (e.g., Coumadin, Rivaroxaban) and Lidocaine 
allergy/sensitivity are exclusion criteria for the biopsy procedure 
Before performing the tissue biopsies, Dr. Haus (or his qualified co-investigator who is 
performing the biopsies) will: 
1) Confirm the participant’s identification 
2) Thoroughly explain the biopsy procedures to the participant, describing what they 
may experience during the procedures. 
3) Confirm the location of the procedures (e.g., left or right thigh for muscle biopsy) 
Skeletal Muscle Biopsy Procedure  
• Confirm that the participant does not have any known allergies/sensitivity to local 
anesthetic (e.g., Lidocaine/Xylocaine/bupivacaine) – this should also have been 
addressed before/during informed consent process 
• If necessary, shave ~5” x ~5” square above the vastus lateralis 
• Put on surgical gown, mask and bonnet (assistants in procedure room also must 
wear mask and bonnet) 
• Disinfect skin above the vastus lateralis with betadine or chlorohexadine 
• Put on sterile gloves and all the following procedures must be performed using 
aseptic technique 
• Create sterile field on tray top and around the antiseptic covered area of the 
participant’s thigh  
• Infiltrate skin and underlying tissue with approve analgesic using a 25g x 1.5” or 
similar needle (For example, if using lidocaine ~5-10 ml will be administered: total 
dose of lidocaine not to exceed 4.5mg/kg) 
• Make an incision above the vastus lateralis with a #10 or #11 scalpel (cut skin and 
try to cut fascia if possible) 
• Apply pressure to incision site with sterile gauze until bleeding stops (or is very 
light)  
• While pressure is being applied to the thigh, the biopsy assistant attaches a 3-way 
stopcock to a 60cc or similar syringe 
• One end of sterile pressure tubing is attached to the sterilized biopsy needle (4-5g 
Bergstrom or UCH biopsy needle), and the other end of the tubing is handed to the 
assistant to attach to their 3-way stopcock to syringe (or similar) set-up for suction 
• The biopsy needle is inserted into the incision with firm pressure, the biopsy needle 
is inserted past the fascia, in the location/direction of the applied anesthetic  
• Once the biopsy needle is fully inserted, the biopsy assistant pulls back firmly on 
the plunger of the syringe to apply steady suction 

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19 
• The sample is collected by closure of the biopsy needle – To increase sample 
yield, the biopsy needle can be quickly rotated then opened and closed 3-5 times 
to obtain a few “snips” of sample before removing the biopsy needle from the thigh 
• The sample is placed on a sterile absorbent pad, and pressure is applied to the 
biopsy site with sterile gauze  
• The biopsy assistant quickly rinses/cleans the sample and gently dabs the sample 
on the absorbent pad to remove excess liquid 
• The sample is processed according to PI discretion 
• While research team member is cleaning and storing the sample, manual pressure 
is applied to the incision site 
• The biopsy procedure can be repeated in the same incision site (or separate pre-
prepared site) if more tissue is needed  
• When the entire biopsy procedure is finished, firm direct pressure is applied to the 
incision site until bleeding stops (~10 minutes) 
• Incision site is cleaned with 70% ethanol and dried with sterile gauze 
• The incision is closed with steri-strips or equivalent and an overlaying transparent 
dressing  
• A pressure dressing is applied to the thigh until bedtime the night of the biopsy 
procedure 
• Participant is instructed to avoid water submersion for 3 days and to keep the 
bandages on for 3-5 days 
Upon discharge, provide participant with pre-printed post-procedure care instructions with 
emergency contact information. 
Acute Aerobic Exercise Bout: Treadmill Walking 
Subjects will complete an acute aerobic exercise bout coupled with muscle 
biopsies and adipose microdialysis at baseline and following the 12-week intervention 
period. This exercise bout includes 60 min of treadmill walking at ~65-70% of their 
predetermined VO2max (VO2max will be measured approximately one week prior to the 
acute exercise bout). The acute exercise bout is considered “moderate” intensity, given 
the relatively low heart rate maintained and muscles utilized. Subjects will be familiarized 
to the protocol before testing. For all acute exercise trial sessions, heart rate and oxygen 
consumption will be monitored to assess the exercise intensity. The acute aerobic 
exercise bouts will be performed at MCRU or Domino Farms and supervised by the 
research team which will include a study physician on-call. 
 
 
 
 
 

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20 
Microdialysis 
Our lab has experience with the microdialysis technique and has published muscle 
specific microdialysis data with acute aerobic exercise in human subjects [48]. We have 
also enlisted the expertise of Dr. Robert Hickner, PhD (Florida State University) to provide 
consultation on microdialysis best practices and interpretation of data generated. Dr. 
Hickner is a microdialysis expert 
who is one of the original 
developers of the microdialysis 
methodology in skeletal muscle 
nearly 25 years ago [49].  
 
Microdialysis 
catheters 
mimic blood capillaries (Figure 
4). 
Substances 
from 
the 
extracellular space can be 
collected into the catheter. The 
timeline of microdialysis events 
are portrayed in Figure 5. Two, 
small, M Dialysis 71 high cut-off 
microdialysis catheters will be 
inserted into the abdominal 
subcutaneous adipose bed (no 
closer 
than 
3 
in 
to 
the 
umbilicus). The microdialysis 
probes will be perfused with 
sterile water at 0.3 µl/min for 10 
minutes before and immediately 
after probe insertion with a calibrated micro infusion pump. The probes will then be 
disconnected from the perfusion pump and left in place with the inlet and outlet tubing 
capped, which is secured to the leg overnight. The timing of the probe insertion 
(approximately 12 hr before the resting measurements) is to ensure that the resting 
measurements are not influenced by the probe insertion [50, 51]. The following morning 
each subject will undergo the baseline measurement of resting adipose tissue sRAGE 
collected by the microdialysis probe. The microdialysis probes are perfused with a sterile 
solution supplemented with 37 g/L dextran-70. Dextran is used to assure there is no net 
fluid transport across the microdialysis membrane [52, 53]. Therefore, the subjects do not 
come into contact with the dextran solution. Samples will be collected in sealed microvials 
that are pre-weighed before and after each collection period to determine actual dialysate 
weight and calculate dialysate volume. At the start of each microdialysis measurement, 
each probe is reconnected to the perfusion pump and perfused with sterile water for 10 
minutes at 0.3 µl/min and then at 0.3 µl/min for 3 hr while the subjects rest quietly or 4 hrs 
post-acute aerobic exercise.  Subjects will be instructed on how to care for the wound 
and supplied with ice and pressure bandages after to reduce inflammation and any 
soreness. 
Figure 4 
Courtesy of MDialysis 

 _____________________________________________________________________________________________ 
21 
 
 
 
Initial Exercise Bout (moderate treadmill walking) 
Upon completion of the baseline testing period, all subjects will be asked to 
undergo a single, modest treadmill aerobic exercise bout (65% VO2max for 30 minutes). 
Prior to this trial, each participant will have been medically cleared for exercise based on 
screening criteria. This treadmill bout will be supervised by a member of the research 
team and may be completed at CCRB, Domino Farms, or MCRU (or other appropriate 
University facility). Participants will be familiarized with the protocol, In addition to 
intermittent oxygen consumption evaluation, subjects will be fitted with a heart rate 
monitor to track exercise intensity. Each subject will have a simple blood draw before and 
after the trial (~80 ml total). This exercise will count as the first exercise training session 
for subjects randomized to the training group and subjects randomized to the Control 
group will be advised to resume normal daily activity and standard of care for the duration 
of the study. 
 
 
Follow-up Testing 
All subjects will return for follow-up testing at approximately weeks 4 and 8. Each 
subject will have a blood draw (~20 ml) and the AE group will also complete a VO2max 
test to adjust target HR for the training protocol.  
 
Final Study Visit Testing 
The final study visit at MCRU or Domino Farms will include the same testing 
procedures for both groups that were completed during baseline testing (i.e., body 
-12hr
0
2hr          4hr           6hr
8hr
Probe insertion
Microdialysis collection
sRAGE measurement via microdialysis 
sRAGE
 
 
EX
sRAGE
X 
Probe insertion 
sRAGE measurement via microdialysis 
Muscle biopsy 
Acute aerobic exercise trial 
Figure 5 
4hr 
6hr 

 _____________________________________________________________________________________________ 
22 
composition assessment, cognitive assessment, OGTT, measurement of insulin 
sensitivity). In addition, all subjects will undergo an acute aerobic exercise bout (treadmill 
walking) coupled with microdialysis and muscle biopsies.  
 
Total Study Tissue Sampling 
Table 1 outlines the approximate amount tissue to be collected during the study. 
Tissue samples (blood, urine, and muscle) will be obtained during the screening and/or 
baseline (before the 3 month AE intervention or control period), follow-up visits, and final 
study visits (after the 3 month AE intervention or control period) time points. Therefore, 
these time points will be separated by >3 months. In addition, participants in this study 
will be overweight or obese and have a body weight > 110 lb. Thus, the proposed blood 
draws are acceptable by University of Michigan IRBMED guidelines. 
 
 
 
 
 
 
Sample Preparation, Storage, and Shipping 
Study samples and resultant data will not contain personal identifying information. 
Each subject will receive a unique study identifier and samples will be appropriately 
labeled based on the testing procedure and sample type. All source document data will 
be kept in a locked file cabinet and converted to electronic data. All electronic data will be 
safe-guarded by password protection on a University of Michigan server. Sample 
preparation will be completed by the research team and/or MCRU/NORC staff during 
each procedure. This includes processing of muscle biopsy, blood, and microdialysis 
Table 1 
 
 
Time point 
Blood 
Muscle 
Screening Visit 
 
 
Both groups 
20 ml 
- 
Baseline Visit 
AE group 
280 ml 
400 mg 
Control group 
280 ml 
400 mg 
Initial Exercise Bout 
 
 
AE group 
80 ml 
- 
Control group 
80 ml 
- 
Follow-up Visits 
 
 
Both groups 
40 ml 
- 
Final Visit 
AE group 
280 ml 
400 mg 
Control group 
280 ml 
400 mg 
AE group total 
~700 ml 
~800 mg 
Control group total 
~700 ml 
~800 mg 

 _____________________________________________________________________________________________ 
23 
samples. Samples will be stored in a -80 °C freezer. Handling and shipping of study 
samples will only occur by the trained research staff as directed by Michigan Occupational 
Safety and Health Act (MIOSHA) and Occupational Safety and Health Administration 
(OSHA) guidelines. 
 
Assessment of Safety 
The risk to benefit ratio for this study is excellent. Overall, the risks are minor with 
the information gained on the impact of exercise on diabetes invaluable. Safety will be 
regularly monitored through personal communication, observation, and documentation. 
Each individual involved in this study will be encouraged to communicate needs and any 
potential issues that arise. 
 
Adverse Events 
Members of the research team will be present for all components of the study to 
monitor safety. Additionally, the research team will communicate weekly for review of 
safety and data collection. In the situation of an adverse event, the event will be 
immediately managed an IRBMED Adverse Event Report Form will be sent to the 
appropriate clinical site and Institutional Review Board in a timely manner. This report will 
include a full description of the event, including the relationship of the incident to the test 
procedure.  
 
Potential Adverse Events and Mitigation Strategies 
Breach of Confidentiality: There is a potential for breach of confidentiality. Study 
samples and resultant data will not contain personal identifying information. Each sample 
will be stored with a unique code. All source document data will be kept in a locked file 
cabinet and converted to electronic data. All electronic data will be safe-guarded by 
password protection and encryption on a University of Michigan server. 
 
Muscle Biopsy: Risks include discomfort, infection, or excessive bleeding at the biopsy 
site. These complications will be minimized using sterile techniques and pressure by an 
experienced team member. Subjects will be instructed that they should report any 
bleeding or opening of the wound immediately for follow up care. Subjects will be supplied 
with ice and pressure bandages after each biopsy to reduce inflammation and any 
soreness after the biopsy. A member of the research team will follow-up with participants 
the day after each muscle biopsy. 
 
Lidocaine: Lidocaine is a local anesthetic administered during the muscle biopsy 
technique. It is possible to develop an allergic reaction to lidocaine, which could range 
from minor itching and a rash to severe respiratory arrest and death. This is a possibility 
with all medications and we will include subjects in the study only if they are without a 
history of an allergic reaction. Medical treatment will be provided if an allergic reaction is 
evident. 
 

 _____________________________________________________________________________________________ 
24 
Bupivacaine: Bupivacaine is a local anesthetic delivered during the muscle biopsy 
technique. It is possible to develop an allergic reaction to bupivacaine, which could range 
from minor itching and a rash to severe respiratory or cardiac arrest and death. This is a 
possibility with all medications and we will include subjects in the study only if they are 
without a history of an allergic reaction. Medical treatment will be provided if an allergic 
reaction is evident. 
 
DEXA: There is a risk of x-ray exposure with DEXA. The duration of each scan will be 
less than 10 min and the subject will be exposed to a radiation dose of ≤ 0.3 mrem based 
upon manufacturer’s specifications and calculations from Stanford Dosimetry, LLC 
RADAR Medical Procedure Radiation Dose Calculator. This amount of radiation exposure 
is considered “general public” exposure by the Nuclear Regulatory Commission. 
 
Blood Samples: Inserting a needle for blood sampling may be associated with some 
discomfort, bruising, and on a very rare occasion, with inflammation. Strict sterile 
procedures will be utilized during blood draws to minimize risks. 
 
Urine Collection: There are no known risks to collecting urine samples. However, some 
people may find it uncomfortable or embarrassing to collect samples. 
 
EKG: Men may need to have a small amount of chest hair shaved off for the ECG pads 
to stick correctly. A small amount of adhesive might remain on the skin when the pads 
are removed or the adhesive might pull on hair when removed. 
 
Exercise: There is a risk of physical injury or fatigue due to the cardiorespiratory fitness 
test and participation in an exercise intervention. However, these risks are generally 
considered rare and minor. Our investigation team has extensive experience conducting 
graded exercise tests in a number of special populations (including elderly and diseased 
populations) and is well versed in how best to minimize risk during these tests. In addition, 
exercise testing and training will be performed in a facility equipped with AED devices 
and staff trained in CPR. 
 
Insulin Clamp Procedure: There is a risk that the subject may react to the infusion of 
glucose or insulin. These reactions could include low blood sugar, an increase in blood 
pressure, flushing and/or sweating. In the event one of these reactions occur, medical 
staff will be notified and appropriate measures will be taken. 
 
Heated Hand Box: There is a minor risk of skin discomfort or burn. If the temperature is 
too warm it can be reduced to improve comfort. 
 
Oral Glucose Tolerance Test: There is a minor potential risk of reaction to the ingestion 
of glucose. These reactions could include nausea, low blood sugar, an increase in blood 
pressure, flushing and/or sweating. Continuous blood glucose monitoring will take place 
during this procedure to provide real time feedback of glucose levels. 
 
Microdialysis: Inserting the microdialysis probes may be uncomfortable or painful. 

 _____________________________________________________________________________________________ 
25 
Study Diet: It may be burdensome for subjects to adhere to a special diet for an extended 
period of time. The metabolic kitchen staff at MCRU will work to accommodate participant 
needs. 
 
Unforeseeable Risks: There may be risks or side effects related to the study that are 
unknown at this time. Continuous monitoring of all participants will facilitate appropriate 
action in the event of an unforeseeable risk. 
 
Expected Adverse Reactions 
There are no expected adverse events. 
 
Serious Adverse Events and Unanticipated Problems 
Subjects will be closely monitored by the research team, MCRU, CVC, and/or 
Domino Farms staff during all testing procedures. Should a serious event occur, it will be 
managed promptly by the research team and appropriate medical staff. 
 
Abnormal Laboratory Test Values or Abnormal Clinical Finding Procedure 
Should an abnormal test value or finding be identified, the subject will be promptly 
notified and encouraged to communicate the finding with their personal physician.  
 
Reporting Procedures 
All adverse events will be reported to the research team for awareness. The study 
principal investigator, Dr. Jacob Haus PhD, and a study physician, will ensure the 
University of Michigan Institutional Review Board is notified within a timely manner. 
 
Safety Oversight 
Dr. Jacob Haus, PhD and Dr. Elif Oral, MD (the physician-of-record) will provide 
safety oversight for the study. Dr. Jeffery Horowitz, PhD (School of Kinesiology) will serve 
as our data safety monitor.  
 
 
STATISTICAL CONSIDERATIONS 
Study Hypotheses 
Our central hypothesis is that skeletal muscle contributes to sRAGE appearance 
in the circulation and maintenance of healthy levels of total sRAGE promotes 
cardiometabolic health. Therefore, two specific working hypotheses have been proposed. 

 _____________________________________________________________________________________________ 
26 
1) Skeletal muscle expression of RAGE will decrease following 12-weeks of aerobic 
exercise training and this change will be related to increases in insulin sensitivity 
and cardiorespiratory fitness. 
2) Circulating sRAGE levels will increase following 12-weeks of aerobic exercise 
training and this change will be related to reduced muscle RAGE expression and 
increases in insulin sensitivity and cardiorespiratory fitness. 
 
Statistics and Sample Size Considerations 
The primary outcomes of this study involve collecting data at pre-aerobic exercise 
training and 12 weeks of training. Comparisons of trajectories across groups (training vs. 
Control) over time are made with appropriate adjustments for fixed (age, sex, 
medications, diabetes duration and severity) and time-varying covariates (i.e., weight, 
BMI, etc). Because of the anticipated nonlinear response curves of outcome variables we 
will use nonlinear mixed effects models to analyze our data. Using PROC NLMIXED in 
SAS 9.2 (SAS Inc.) we will fit the nonlinear models using two likelihood-based methods: 
adaptive Gaussian quadrature and a first-order Taylor series approximation [54-56]. The 
nonlinear mixed model is: y = f(xij, β, ui) + eij, where f is some nonlinear function of known 
vector covariates (xij) for the jth observation on the ith subject, unknown fixed effect 
parameters (β), an unknown vector of random effect parameters (ui), and unknown 
random errors (eij). For this study, we will treat training group as main fixed effect, 
intercept as random effects, and all outcomes will be used as responsive variables. Using 
nonlinear mixed effects models described above, we will reject null hypothesis if 
significant (p<.05) interaction effect between group and time is observed. Post-hoc 
analysis will examine differences between groups at different time points to assess the 
effect of exercise training on the outcome variables across time during the intervention. 
Our human sample size was determined using data from preliminary studies to provide 
adequate power after attrition to test the hypothesized effect of training on skeletal muscle 
RAGE and sRAGE concentrations. We will enroll 75 subjects with the anticipation that 
there will be 20-25% attrition at 12 weeks. We anticipate that the final N after attrition will 
be 60 (n= 30 per group). Using the two-sided alpha level of 0.05, we estimate that a total 
sample size of 60 will enable us to detect a pre-post, between-group difference of 1.24 
standard units for RAGE between Aerobic Exercise training and Control groups with a 
target of 90% statistical power.  
 
Safety and Efficacy Review 
Our team has a Michigan Occupational Safety and Health Act (MIOSHA) approved 
chemical hygiene plan in place. This hygiene plan requires extensive training in the 
University of Michigan’s Responsibility Conduct of Research training program, known as 
PEERRS (Program for Education and Evaluation in Responsible Research and 
Scholarship), first aid, and CPR.  The research team will be meet weekly to discuss study 
progress, training, and ensure study objectives and guidelines are met. 
 
 

 _____________________________________________________________________________________________ 
27 
Analysis Plan 
Due to nature of this study, data will be analyzed on an on-going basis. Participant 
enrollment will be rolling with sample collection occurring intermittently. Samples will be 
batched and analyzed as appropriate. Study samples and resultant data will not contain 
personal identifying information (only a unique study code). All source document data will 
be kept in a locked file cabinet and converted to electronic data. All electronic data will be 
safe-guarded by password protection on a University of Michigan server. 
 
 
QUALITY CONTROL AND QUALITY ASSURANCE 
 
Ethics and Protection of Human Subjects 
Coupled with extensive experience testing human subjects in a variety of settings, 
the training provided by the University of Michigan’s Responsibility Conduct of Research 
training program, known as PEERRS (Program for Education and Evaluation in 
Responsible Research and Scholarship) will guide the protection of the research team 
and study participants.  
 
Informed Consent Process  
As explained in detail elsewhere, initial screening of participants will occur via a 
website screening tool or telephone interview. During this time, the study will be described 
in detail and we will obtain information about age, body mass index (BMI), smoking and 
physical activity status, current medical conditions, health status and medications. 
Subjects who meet the initial screening criteria are scheduled for a medical screening. 
These tests will be performed in the Michigan Clinical Research Unit (MCRU), CVC, 
Domino Farms, and/or the CCRB (or other University recreation building). Verbal and 
written informed consent will be obtained during this initial visit according to the guidelines 
of our Institutional Review Board. Subjects are screened based on the inclusion/exclusion 
criteria. 
 
Informed Consent/Assent Process (in Case of a Minor) 
Minors will not participate in this study. 
 
Subject Confidentiality 
The screening process utilized in this study was selected to minimize study risk. 
All conversations between the research team and participants are considered 
confidential. Screening will take place in a private environment with only the research 
team and/or Michigan Clinical Research Unit (MCRU), CVC, or Domino Farms staff 
present. Study samples and resultant data will not contain personal identifying 
information. All source document data will be kept in a locked file cabinet and converted 

 _____________________________________________________________________________________________ 
28 
to electronic data. All electronic data will be safe-guarded by password protection on a 
University of Michigan server. 
 
Study Discontinuation 
Participants are free to withdraw their consent and discontinue participation at any 
time. We do not anticipate participant withdrawal due to study-related reasons as similar 
exercise training interventions have been taking place for decades with high levels of 
success. Additionally, we have experience managing clinical trials in the proposed 
population [26, 30, 34, 35]. However, we recognize participants interests, health and 
schedules may change. The research team’s best effort will be made to accommodate 
the participants needs and minimize attrition. 
 
Future Use of Stored Specimens 
Participants will be asked to consent for future use of their samples. We expect 
that the data and tissue and blood samples obtained from the proposed study will have 
lasting research value to maximize productivity for the research team.  We intend to use 
these resources to explore future mechanisms and the data obtained that can be mined 
for new and impactful opportunities. 
 
DATA HANDLING AND RECORD KEEPING 
Data Management Responsibilities 
 Data will be managed internally by the research team. Source study documents 
will be converted to electronic files as needed and interpreted by the research team on 
an on-going basis. The source study documents will be safe-guarded in a locked file 
cabinet. Weekly study team meetings will address data management needs. 
 
Data Collection Process 
Data collection will be conducted by the research team, MCRU, CVC, and/or 
Domino Farms staff at the University of Michigan. Specifically designed protocol 
documentation forms will guide the data collection process.  
 
Study Records Retention 
Similar to the future use of stored samples, we intend to use study records as 
resources to explore future mechanisms and the data obtained that can be mined for new 
and impactful opportunities. Study records provide important clinical context and 
interpretation of study findings.  
 

 _____________________________________________________________________________________________ 
29 
Protocol Deviations 
Study protocol deviations are not anticipated. In the event a deviation from study 
protocol becomes necessary, documentation and dissemination within the research team 
and to the Institutional Review Board (as needed) will occur in a timely fashion. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 _____________________________________________________________________________________________ 
30 
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Galichet, A., M. Weibel, and C.W. Heizmann, Calcium-regulated intramembrane 
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Metz, V.V., et al., Induction of RAGE shedding by activation of G protein-coupled 
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