Effects of Pioglitazone on Exogenous Carbohydrate Oxidation During Steady-State Exercise at High Altitude
Phase: PHASE4
Conditions: High Altitude
Sponsor: United States Army Research Institute of Environmental Medicine

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 1 of 26 
PROTOCOL TITLE: Effects of Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State 
Exercise at High Altitude        
 
SECTION A:  RESEARCH TEAM AND LOCATIONS 
 
A1.  RESEARCH TEAM 
 
Study Role 
Institution/Company and Contact Information 
 
Sponsor 
N/A     
 
Principal Investigator 
 Name, Rank, and Degree: Lee M. Margolis, PhD  
Title:  Nutritional Physiologist 
Institution:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2335 
Email:  lee.m.margolis.civ@health.mil 
 
Associate Investigator(s) 
Name and Degree: Stefan M. Pasiakos, PhD 
Title:  Division Chief 
Institution:  MPD, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2250 
Email: stefan.m.pasiakos.civ@health.mil     
 
 
Name and Degree: Roy M. Salgado, PhD 
Title: Research Physiologist  
Institution:  TMMD, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2375 
Email: roy.m.salgado.civ@health.mil   
 
 
Name and Degree: Benjamin J. Ryan. PhD 
Title: Research Physiologist  
Institution:  TMMD, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2408 
Email: benjamin.j.ryan.14civ@health.mil   
 
Name and Degree: Emily E. Howard, PhD 
Title: Nutritional Physiologist  
Institution:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2309 
Email: emily.e.howard14.civ@health.mil   
 
Name and Degree: Jessica A. Gwin, PhD 
Title: Research Physiologist  
Institution:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2300 
Email: jessica.a.gwin.civ@health.mil   
 
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 2 of 26 
Name and Degree: Devin Drummer, PhD 
Title: ORISE Post-Doctoral Research Fellow  
Institution:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2461 
Email:  devin.j.dummer.ctr@health.mil  
 
Name and Degree: Michael A. Dawson, MAJ, PhD, RD, CSSD, 
LD 
Title: Research Dietician  
Institution:  MND, USARIEM 
Address:  10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-206-2278 
Email:  michael.a.dawson62.mil@health.mil  
 
   
Study Consultant 
 
 
 
 
 
 
 
Study Coordinator                           
Name and Degree: Andrew J. Young, PhD 
Title: Senior Nutritional Physiologist 
Institution: MND, USARIEM 
Address: 10 General Greene Ave Bldg. 42, Natick, MA 01760 
Phone Number: 508-353-4160 
Email: andrew.j.young60@gmail.com     
 
 
Name, Rank, and Degree:  Marques A. Wilson, MS 
Title:  Research Physiologist 
Institution/Company:  MND, USARIEM 
Address:  10 General Greene Ave, Bldg 2, Natick, MA 01760 
Phone Number:  508-206-2411 
Email:  marques.wilson.civ@health.mil  
 
Research Monitor 
 
 
 
 
 
 
Ombudsman  
 
Name, Rank, and Degree: Sarah L. Hanson, MAJ, DO 
Title: Physician 
Institution/Company: OMSO, USARIEM 
Address: 10 General Greene Ave Bldg. 42a, Natick, MA 
Phone Number: 508-206-2460 
Email: sarah.l.hanson3.mil@health.mil 
 
Name, Rank, and Degree:  Adam Luippold, BS 
Title:  Research Physiologist 
Institution/Company:  TMMD, USARIEM 
Address:  10 General Greene Ave, Bldg 2, Natick, MA 01760 
Phone Number:  508-206-2330 
Email:  adam.j.luippold.civ@health.mil  
 
Name, Rank, and Degree: Andrew Greenfield, MS 
Title: ORISE Research Fellow 
Institution/Company: TMMD, USARIEM 
Address: 10 General Greene Ave, Bldg 2, Natick, MA 01760 
Phone Number: 508-206-2474 
Email: andrew.m.greenfield.ctr@health.mil 
 
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 3 of 26 
A2.  ROLES AND RESPONSIBILITIES 
      
       A2.1 Principal Investigator   
 
Name:  Lee M. Margolis 
Study Responsibilities: Dr. Margolis is responsible for the safe and scientifically sound conduct of the 
study.  He will oversee all aspects of the study, ensure safety and ethical treatment of volunteers; 
maintain required documentation for the study and obtain required approvals; and will have primary 
responsibility for data analysis, interpretation, and publication. Dr. Margolis will also be actively involved 
in the volunteer brief, obtaining consent, and data collection. Dr. Margolis will be responsible for the 
conduct of the study in accordance with the protocol.  Maintenance of a list of appropriately qualified 
persons to whom significant study-related responsibilities have been delegated.   
 
        A2.2 Associate Investigator 
 
Name(s):  Stefan M. Pasiakos, Roy M. Salgado, Benjamin J. Ryan, Emily E. Howard, and Jessica A. 
Gwin, Devin Drummer, Michael Dawson      
Study Responsibilities:  Protocol development, formulation of protocol questions, hypotheses, 
experimental approach, and design. Assist PI with volunteer briefing, obtaining informed consent, data 
collection, and sample processing. Associate investigators will also ensure safety and ethical treatment 
of participants and will contribute to the analysis, interpretation, and publication of the data.        
 
       A2.3 Study Coordinator 
 
        
Name:  Marques Wilson 
Study Responsibilities:  Primary responsibilities include assisting with volunteer briefing, obtaining 
informed consent, data collection, preparing and administering study diets, preparing and maintaining 
data collection forms, and biological sample processing. Will contribute to the analysis, interpretation, 
and publication of the data.  She may conduct procedures for which she is qualified and credentialed as 
outlined in the DOA log.   
 
        A2.4 Ombudsperson   
 
            Name(s): Adam Luippold and Andrew Greenfield      
Study Responsibilities: The ombudsman shall protect the interests of the research volunteers. The 
ombudsman will do the following: read the consent form, observe the recruitment briefing and the 
signing of consent documents, make sure supervisory and command staff are not present, make sure 
the description of the study and all risks and discomforts are correct, complete, and understandable to 
the audience, and make sure that the volunteers are not threatened or pressured to participate.  They 
will observe briefings for military participants in the Human Research Volunteer (HRV) program.      
 
A2.5 Study Consultant 
 
Name(s): Andrew J. Young 
Study Responsibilities:  Study consultant will provide insight and feedback on study design and data 
interpretation. Activities may include protocol development, formulation of protocol questions, 
hypotheses, experimental approach, and design. They will not engage with participants or assist with 
data collection tasks.  
 
A2.6 Research Monitor 
Name(s): Sarah L. Hanson 
Study Responsibilities: The research monitor will review reports of SAEs/UPIRTSOs and provide an 
unbiased written report of the events. The research monitor should periodically observe the conduct of 
the study with subjects.  The research monitor should provide concurrence that subjects met all 
protocol eligibility criteria for the study.  

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 4 of 26 
 
 A3.  RESEARCH LOCATIONS    
 
USARIEM, Natick MA: The U.S. Army Research Institute of Environmental Medicine (USARIEM) is a 
DoD research facility within the U.S. Army Medical Research and Development Command.  It is the 
Institute responsible for conducting basic and applied research to determine the effects of exposure to 
environmental extremes, occupational tasks, physical training, deployment, operational stress and 
nutritional factors on the health and performance of military personnel. The facility contains 
environmental chambers for controlling temperature and humidity, an environmentally controlled 
hypobaric chamber, a water immersion laboratory, as well as several dry and wet laboratories for 
animal and human experimentation. The dry laboratories are capable of a broad range of experiments, 
including biomechanical analysis, body composition, energy expenditure, muscle strength and 
endurance. The wet laboratories include general clinical chemistry analyzers, as well as equipment for 
ELISA, RIA, histology, and molecular biology assays.  Each investigator at the facility has a personal 
computer with software for data management, analysis, presentation and report generation. Their 
computers are interfaced with a network server for easy, secure data handling and transfer. All testing 
procedures will take place at USARIEM. 
 
Metabolic Solutions, Nashua, NH: Metabolic Solutions is a state-of-the art stable isotope analytical 
laboratory that has the basic laboratory facilities and equipment to analyze stable isotope in breath and 
blood.  Equipment onsite includes an Agilent 6110 LC-Tandem Mass Spectrometer (LC-MS/MS) Triple 
Quad with Agilent 1100 LC, Thermo Finnigan Delta XP Isotope Ratio Mass Spectrometer (IRMS) with 
GC Isolink for 13C, 15N and 2H GC-IRMS and Conflow IV interface, and a Thermo Finnigan Delta V 
Advantage IRMS with trace GC-Combustion III unit for 13C, 15N and D GC-C-IRMS and Conflow IV 
interface and elemental Analyzer EA112HT 13C, 15N and D sample combustion unit. Metabolic 
Solutions has been an industry leader in developing new stable isotope tracer applications.  Metabolic 
Solutions will analyze samples on a fee for service basis. 
 
Pennington Biomedical Research Center (PBRC), Baton Rouge, LA: The laboratory is accredited by 
the College of American Pathologists (CAP) and participates in proficiency testing programs 
administered by the CAP. Biochemical analyses will be conducted at PBRC on a fee for service basis.  
                 
A4.  MULTISITE RESEARCH   
 
Lead Site: N/A  
Performance Site: N/A 
 
 
 
SECTION B:  RESEARCH METHODOLOGY 
 
B1.  ABSTRACT   
Two recent investigations from our laboratory have consistently observed a reduction (30-50%) in the rate of 
exogenous glucose oxidation in native lowlanders performing metabolically-matched, steady-state exercise at 
high altitude (HA) compared to sea level (SL). Accompanying lower rates of exogenous glucose oxidation at 
HA were increases in circulating glucose and insulin concentrations, and reduced glucose rate of 
disappearance and metabolic clearance rate. Apparent hypoxia-induced insulin insensitivity along with 
alterations in glucose kinetics suggests reduction in glucose uptake by the peripheral tissue is a primary factor 
contributing to reductions in exogenous glucose oxidation at HA. As such, the primary objective of this study is 
to determine the ability of an insulin sensitizer (Pioglitazone, PIO) to enhance exogenous glucose oxidation 
and metabolic clearance rate during metabolically-matched, steady-state exercise during acute HA exposure 
compared to placebo (PLA) in native lowlanders. Secondary objective of this study will be to assess the impact 
of PIO on markers of inflammation and iron status compared to PLA. This randomized crossover placebo 
control double blinded study will examine substrate oxidation and glucose kinetic responses to ingesting 
supplemental carbohydrate (glucose) during metabolically-matched, steady-state exercise with acute (~5 h) 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 5 of 26 
exposure to HA (460 mmHg, or 4300m, barometric pressure similar to Pike’s Peak) after receiving PIO 
(HA+PIO), or after receiving a matched placebo (HA+PLA). Eight healthy, recreationally active males between 
the ages of 18-39 yrs will be required to complete this study. Following a 4 day glycogen normalization period 
receiving PIO or PLA daily, volunteers will complete two 80-min trials, performing metabolically-matched, 
steady-state aerobic (same absolute workload corresponding to ~55  ±5% of V̇ O2peak at HA) exercise on a 
treadmill, and consuming 145 g of glucose (1.8 g/min); one trial with HA+PIO and the other with HA+PLA. A 
dual glucose tracer (13C-glucose oral ingestion and [6,6-2H2]-glucose primed, continuous infusion) technique 
and indirect calorimetry will be used to selectively analyze endogenous and exogenous glucose oxidation, as 
well as glucose rate of appearance (Ra), disappearance (Rd) and metabolic clearance rate (MCR). Serial 
blood samples will be collected during each trial to assess endocrine and circulating substrate responses to 
exercise, carbohydrate, and hypoxia with or without PIO. All trials will occur at the same time of day in the 
USARIEM hypobaric/hypoxic chamber and be separated by a minimum 10-d washout period. The primary 
risks associated with this study include those associated with acute hypobaric hypoxia, exercise, and blood 
sampling. 
  
B2.  BACKGROUND AND SIGNIFICANCE   
Acute High Altitude Exposure and Glucose Metabolism 
Deployments to austere and remote mountainous regions will occur during Multi-Domain Operations (MDO). 
Rapid ascent to high altitude (i.e., hypobaric hypoxia; HA) elicits physiologic and metabolic adjustments that 
collectively degrade physical performance and augment dietary requirements. In particular, HA exposure 
increases reliance on carbohydrate for fuel to sustain physical activity, resulting in rapid declines in muscle 
glycogen content which, in part, contributes to decreased performance (1-3). To combat declines in physical 
performance, the Modular Ration Enhancement Operations HA 
(MORE-HA) was created to provide additional dietary carbohydrate 
to fuel sustained physical activity. However, recent studies from our 
laboratory have consistently demonstrated a 30-50% reduction in 
oxidation of exogenous glucose consumed by unacclimatized 
lowlanders performing steady-state exercise during acute (< 8 hrs) 
high altitude (HA) exposure compared to metabolically-matched 
exercise performed at sea level (SL; Figure 1) (3, 4). Reduced 
exogenous glucose oxidation during acute (< 8 hrs) HA exposure 
has also been demonstrated by others (5-7). Across studies, 
reductions in exogenous glucose oxidation occurred whether 
exercise at SL and HA were matched for absolute (3-5) or relative 
(6, 7) intensity. Consistent results between investigations from 
multiple laboratories, matching for different exercise intensities, 
indicates a true physiological dysregulation in use of exogenous 
carbohydrate for fuel use during aerboic exercise after initial arrival 
to HA from SL.       

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 6 of 26 
Accompanying lower rates of exogenous 
glucose oxidation at HA are increases in 
circulating glucose and insulin concentrations 
(Figure 2A,B), and reductions in glucose rate of 
disappearance and metabolic clearance rate 
(MCR; Figure 2C,D) during steady-state 
exercise compared to SL (3). These data are 
characteristic of insulin resistance (8) and 
suggest that reductions in peripheral glucose 
uptake may be a primary factor contributing to 
lower rates of exogenous glucose oxidation at 
HA. The inability to effectively use dietary 
carbohydrate as a metabolic fuel source and the 
likelihood that peripheral insulin resistance 
modulates those effects, suggests that a more 
aggressive solution than carbohydrate 
supplementation alone is required to optimize 
fuel utilization during hypoxia and enhance 
physical performance during future HA MDO.  
 
One possible solution is to employ short-term use of insulin sensitizing drugs, such as biguanides (Metformin) 
and thiazolidinediones (Pioglitazone; PIO), which are commonly prescribed to maintain glucose homeostasis in 
type II diabetics. In the context of acute HA exposure, there is evidence that Metformin may improve hypoxia-
induced insulin sensitivity and increase muscle glycogen synthesis at rest (9). Though Metformin enhances 
insulin sensitivity and peripheral glucose uptake, it also reduces intestinal glucose absorption and hepatic 
gluconeogenesis. The latter effects may limit the amount of exogenous and endogenous glucose available to 
fuel performance. Conversely, PIO’s primary mechanism of action is to target peripheral tissue (e.g., muscle 
and lipid), as well as the liver to improve insulin sensitivity and glucose uptake without affecting intestinal 
glucose absorption. The mechanism of action for PIO suggests it is more appropriate than Metformin as an 
insulin sensitizer to enhance glucose uptake and oxidation to sustain physical performance during 
unacclimatized HA exposure. To date, no study has assessed the effectiveness of PIO on improving insulin 
sensitivity at HA to enhance exogenous glucose oxidation or glucose turnover. 
 
Acute High Altitude Exposure, Inflammation and Iron Status  
Recently, Hill et al.(10, 11) reported that following prolonged exercise (60 minutes at 65% sea level V̇ O2peak) 
in normobaric hypoxia (FIO2 = 0.135 or ~3,500 m) there is an increase in gastrointestinal permeability, 
decrease inflammatory response of peripheral blood mononuclear cells (PBMC), and reduction in the ratio of 
pro- to anti-inflammatory circulatory biomarkers when compared to sea level. From their findings, the authors 
concluded that a short bout of exercise in normobaric hypoxia caused acute immune-disruption. An intriguing 
finding from their study(10) was a decrease in phosphorylated AMP-activated protein kinase (p-AMPK) one 
hour post exercise and a decrease in sirtuin 1 (SIRT1) one and four hours post exercise while simultaneously 
having greater reduction in nuclear factor kappaB (NF-κΒ) one hour post exercise in hypoxic conditions. Both 
p-AMPK and SIRT1 inhibit NF-κΒ inflammatory pathway (12, 13). Thus, the finding of reduced PBMC 
inflammatory response post exercise in normobaric hypoxia seems contradictory.  
 
In addition to its insulin sensitizing effects, PIO has been shown to have anti-inflammatory and antioxidative 
effects. Zhang et al.(14) reported that PIO lowered circulating inflammatory markers in individuals with 
impaired glucose tolerance. PIO has been shown to increase p-AMPK(15) and SIRT1(16) and thus reduce 
inflammation. However, no studies have investigated the effects of PIO on inflammatory responses during 
acute exercise in hypobaric hypoxia particularly when normal health individuals have higher blood glucose 
levels.  
 
An accumulating body of evidence indicates key interactions between hypoxia, insulin resistance, and systemic 
iron homeostasis (17, 18). A secondary effect of improving hypoxia-induced insulin resistance may also be an 
improvement in iron status. Indeed, several cellular mechanisms regulating iron homeostasis are influenced by 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 7 of 26 
PIO-targeted signaling pathways (19, 20). These data may suggest that alleviation of hypoxia-induced insulin 
resistance may also improve iron status. As iron is vital to the oxygen carrying capacity of the blood, improved 
iron status under hypoxic conditions may facilitate improve performance during unacclimatized military 
operations. However, the influence of PIO on systemic iron regulation during acute HA exposure has not been 
determined. 
      
 
B3.  MILITARY RELEVANCE    
Rapid deployment to remote mountainous regions will occur during MDO. Unacclimatized Service members 
experience altered carbohydrate metabolism, causing an increased reliance on glycogen for fuel, concomitant 
with a blunted ability to take up and oxidize exogenous carbohydrate. These negative physiological effects 
likely exacerbate declines in physical performance at HA. This proposal directly supports the Military 
Operational Medicine Research Program’s Physiological Health and Performance Program Area under the 
Biomedical Performance Enhancement (BPE) Work Unit. Specifically, the proposed work aligns to BPE 
Strategic Plan’s Technical Objective 1.1.3: Material solutions (pharmacological) that enhance Soldier 
resistance to environmental stressors. 
This proposal will provide guidance for use of PIO to enhance exogenous carbohydrate oxidation at HA 
operations, and will provide the opportunity to determine if carbohydrate supplementation can indeed enhance 
performance if substrate metabolic dysregulation is avoided during HA exposure. Information from this 
proposal would be valuable to operational partners such as USSOCOM and the 10th Mountain Division who 
engage in HA MDO.  
 
B4.  OBJECTIVES/SPECIFIC AIMS/RESEARCH QUESTIONS  
 
Objective: 
1. Determine the effects of PIO on exogenous glucose oxidation and glucose turnover during exercise under 
acute HA exposure compared to PLA. 
2. Determine the effects of PIO on inflammation during acute HA exposure compared to PLA. 
3. Determine the effects of PIO on markers of iron status during acute HA exposure compared to PLA. 
 
Hypotheses: 
1. Exogenous glucose oxidation, Rd, and MCR during metabolically-matched, steady-state exercise at HA 
will be greater after PIO administration compared to PLA. 
2. Inflammation will be lower during acute HA exposure after PIO administration compared to PLA. 
3. Markers of iron status will be altered during acute HA exposure after PIO administration compared to PLA. 
 
B5.  RESEARCH PLAN   . 
 
       B5.1  Research Design   
This study will be a randomized crossover double blind placebo control trial. 
      
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 8 of 26 
Table 1: Study Timeline 
 
Baseline 
Trial 1 
Washout 
Trial 2 
Study Day 
SV 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10-19 
20 
21 
22 
23 
24 
Medical Screening 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Body Composition 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
VO2peak 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
High Altitude Exposure 
 
 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
Practice Exercise 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
Glycogen Normalization 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
PIO/PLA 
 
 
 
 
 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
Study Diet 
 
 
 
 
 
X 
X 
X 
X 
 
 
X 
X 
X 
X 
 
Steady-State Exercise 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
Carbohydrate Tracer Study 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
Blood Sampling 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
Breath Sampling 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
Timeline may shift based on participant availability, weekend, and holiday schedules. 
 
      B5.2  Research Subjects/Population(s)    
 
                B5.2.1  Subject Population(s)   
 
Subject population will be representative of active duty male service members, being in good health 
and recreationally active. Previous studies at USARIEM (Protocol# 16-02HC and 18-09H) which have 
evaluated metabolically-matched exercise at HA compared to SL have only been performed in male 
volunteers. It is apparent that sex-based differences exist during low-to-moderate intensity endurance 
exercise at sea level. Sex-based differences in substrate oxidation during exercise at SL may result in 
differences in response to change in substrate oxidation at HA between men and women. Therefore, 
this study will recruit male volunteers only as sex differences in glucose metabolism at HA are not 
known.  Based on findings from this and our previous studies, a similar series of investigations will be 
conducted in the future to assess the impact that unacclimatized HA exposure on females. 
 
                B5.2.2  Number of Subjects, Records, and/or Specimens   
To complete testing on the 8 volunteers necessary to reach statistical power, we estimate we will 
need to enroll 24 individuals. All screening will stop once complete data has been collected on 8 
volunteers. Records and specimen collection are described in the Research Procedures and Data 
Collection sections. During briefings and consenting potential participants will be informed that even 
though they may be eligible and want to participate, if we are able to obtain enough data from 
preceding subjects, they may not ultimately be tested. These individuals may be recruited from the 
HRV population, civilians, and active duty military personnel (on and off the installation).   
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 9 of 26 
               B5.2.3  Inclusion Criteria 
• 
Men aged 18 – 39 years 
• 
Born at altitudes less than 2,100 m 
• 
Physically active (exercise minimum 2 days per week) 
• 
Have supervisor approval (permanent party military and civilians) 
• 
Willing to refrain from alcohol, smokeless nicotine products and dietary supplement use during 
study periods 
               B5.2.4  Exclusion Criteria 
• 
Born at altitudes greater than 2,100 m 
• 
Musculoskeletal injuries that compromise exercise capability 
• 
Metabolic or cardiovascular abnormalities, or gastrointestinal disorders 
• 
Taking medication that affects macronutrient metabolism and/or the ability to participate in 
strenuous exercise 
• 
Living in areas that are more than 1,200 m, or traveled to areas that are more than 1,200 m for 
five days or more within 2 months of data collection 
• 
Evidence of apnea or other sleeping disorders 
• 
Prior diagnosis of high altitude pulmonary edema or high altitude cerebral edema 
• 
Presence of asthma or respiratory tract infections 
• 
Smoking or vaping 
• 
Taking medications that interfere with oxygen delivery and transport 
• 
Anemia (HCT <38% and HBG <12.5 g/dL) and Sickle Cell Anemia/Trait 
• 
Blood donation within 8 weeks of beginning the study 
• 
Unwilling or unable to consume study diets or foods provided due to personal preference, dietary 
restrictions, and/or food allergies 
• 
Unwilling or unable to adhere to study physical restrictions 
 
      B5.3  Research Procedures   
Research procedures conducted by investigators and support personnel will be in accordance with the 
Delegation of Authority (DOA) Log for Key Research Personnel as described below.  
 
COVID-19 risk mitigation: Study staff and participants will comply with all COVID-19 risk mitigation 
procedures in place at USARIEM during the time of data collection.  As such, participants may be asked to 
wear face masks and use hand sanitizer during data collection activities (in accord with prevailing 
recommendations at the time of data collection) and may be asked to wear gloves (i.e., nitrile gloves) 
during data collection activities. Study staff and participants also may be asked to undergo COVID-19 
testing (via nose swab performed at USARIEM) before conducting data collection activities and study 
participation in the HA chamber. Participants will be asked if they are experiencing symptoms of COVID-19 
each day. If a volunteer is experiencing or reports symptoms, they will be restricted from study activity, 
including entering the altitude chamber and sent to Office of Medical Support and Oversight (OMSO) for 
further evaluation. Depending on the timing of symptom onset/positive COVID-19 results, the participant 
may be allowed to repeat study activities when symptoms/illness subside.  
 
This randomized crossover placebo controlled double blinded study will examine substrate oxidation and 
glucose kinetic responses to ingesting supplemental carbohydrate (glucose) during metabolically-matched, 
steady-state exercise with acute (~5 h) exposure to HA (460 mmHg) after short-term (5 days) use of 
Pioglitazone (HA+PIO), or matched placebo (HA+PLA). Eight healthy, recreationally active males between 
the ages of 18-39 yrs will be enrolled. Following a muscle glycogen normalization period, volunteers will 
complete 80-min of metabolically-matched, steady-state (same absolute workload corresponding to ~55  ±
5% of V̇ O2peak at HA) exercise on a treadmill, and consume 145 g of glucose (1.8 g/min) with HA+PIO and 
HA+PLA. A dual glucose tracer (13C-glucose oral ingestion and [6,6-2H2]-glucose primed, continuous 
infusion) technique and indirect calorimetry will be used to analyze endogenous and exogenous glucose 
oxidation, as well as glucose rate of appearance (Ra), disappearance (Rd), and MCR. Serial blood 
samples will be collected during each trial to assess endocrine and circulating substrate responses to 
exercise, carbohydrate, and hypoxia, with or without PIO. Isotope methodology and aerobic exercise 
protocols will be identical to our previous work (3, 4), allowing comparison of outcomes across studies. All 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 10 of 26 
trials will be conducted in the USARIEM hypobaric/hypoxic chamber and be separated by a minimum 10-d 
washout period. 
 
A member of the research team will generate a randomization scheme using a random number generator 
(http://randomization.com or similar). Treatment type (PIO/PLA) will be coded to de-identify treatment 
(Example: treatment A and treatment B or similar) to blinded staff. Assigned unblinded staff, responsible for 
administering the treatment to volunteers, will be responsible for creating treatment codes to match 
treatment. They will maintain record of randomization scheme and treatment.    
 
Body Composition: Body composition will be determined using dual energy x-ray absorptiometry (DEXA, 
DPX-IQ, GE Lunar Corporation, Madison, WI). The DEXA technique allows for the non-invasive 
assessment of soft tissue composition by region with a precision of 1-3% (21). The volunteer will lay face-
up on the DEXA densitometer table in shorts, t-shirts, and stocking feet. Volunteers will be asked to remain 
motionless for the 8-10 min scan. These data will be used to calculate total body mass, fat-free mass, and 
fat mass. Calibration to external standards will be performed before actual data collection.  Measurements 
of body composition will be used for participant characterization and to assess if fat mass and fat-free mass 
are confounding variables for substrate oxidation.  
 
Height and Weights: Anthropometrics, performed using standardized techniques and equipment, will be 
used to determine volunteer eligibility and characterize study volunteers. Height will be measured to the 
nearest 0.1 cm using a stadiometer at screening. Body mass will be measured after an overnight fast (10 
hr), using a calibrated digital scale to the nearest 0.1 kg at screening.  Body mass will be measured at 
baseline and the morning of experimental trial days. 
 
Determination of Peak Oxygen Uptake: Following a minimum of a 10 hour fast, volunteers will complete 
two separate maximal aerobic exercise sessions to determine peak oxygen uptake (V̇ O2peak) on a 
treadmill. Two treadmill V̇ O2peak assessments will be conducted because HA exposure of 4,300 m 
produces a decrease in V̇ O2peak of ~27% compared to SL (22).  As such, volunteers will complete one 
treadmill V̇ O2peak at SL and one at HA so V̇ O2peak is known under both conditions.  V̇ O2peak will be 
determined using an indirect, open circuit respirator system (True Max 2400, Parvomedics, Sandy, Utah, 
USA). Volunteers will be clothed in appropriate athletic attire and perform this assessment at standard 
ambient indoor temperature (20-22°C) and humidity conditions (20-30%). Volunteers will be given 
adequate time to become familiar with the testing procedures and allowed a 3-min self-paced warm-up on 
the treadmill. At the start of testing, the volunteer will put on a mask connected to a 2-way respiratory valve. 
The volunteers will begin by running for 4 min at a pace predetermined as comfortable at a 0% grade. At 4-
min, the grade will be increased to 4% followed by an additional 2% every 2 min thereafter until volitional 
exhaustion.  
 
Heart rate will be monitored using a heart-rate monitor (Polar Electro Inc, Oulu, Finland) the last 30 
seconds of each workload during all testing. The test will be stopped immediately if the subject reports 
angina-like symptoms, exertional syncope, shows signs of poor perfusion (i.e., light-headedness, 
confusion, ataxia, pallor, cyanosis, nausea, or cold and clammy skin), or if testing equipment fails. Staff 
conducting this test are trained and certified in basic life support.  
 
Glycogen Normalization: Following an overnight (10 hour) fast, volunteers will complete a glycogen 
normalization protocol on a cycle ergometer. The intensity will be based on V̇ O2peak. Volunteers will begin 
with a 5-min warm-up before beginning the protocol. After a warm-up period, the cycle ergometer protocol 
is comprised of repeated periods of 2 min of work at 80 ±5% V̇ O2peak followed by 2 min of recovery at 50 
 ±5% V̇ O2peak. The protocol will last approximately 50-min (12 work:rest cycles). To ensure familiarity with 
the testing procedures, volunteers will perform one practice session during the baseline pre-study period.  
If volunteers are unable to maintain rotations per minutes (rpm) above 50 for 15 seconds during the work 
stage, watts will be lowered to ensure that the volunteers can complete all 12 work:rest cycles. Volunteers 
will be permitted to consume water ad libitum during the protocol.   
 
Study Diet: After completing the glycogen normalization protocol, volunteers will be fed a controlled diet 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 11 of 26 
prescribed to maintain energy balance for 4 days. The diet will provide 6.0 g carbohydrate/kg/d, 1.2 g 
protein/kg/d, and 1.0 g fat/kg/d.  All food and beverages (except water) will be prepared and provided by 
study dietitians and consist largely of military combat ration and supplemental food items (ex. Frozen 
prepared dinners, breakfast sandwiches, commercial granola bars, etc.). The same diet will be consumed 
for both arms of the study. Volunteers will thus consume the study diet for 8 days total. Corn-derived 
sugars will be avoid in the study diets to reduce high natural abundance of 13C. Caffeine will be restricted to 
no more than one calorie-free caffeinated beverage per day (if the research volunteer chooses to consume 
caffeine). The caffeinated beverage will be supplied by study staff and must not be consumed during 
fasting periods or prior to exercise. Volunteers will be asked to return all wrappers to study dietitians to 
ensure consumption of prescribed study foods.   
 
Insulin Sensitizer: Pioglitazone (PIO) will be administered as a 15 mg oral dose per day for 5 days during 
the HA+PIO arm of the study. A 15 mg dose was chosen because this is the minimal dose required to 
induce a clinically significant improvement in insulin sensitivity (23). Volunteers will take the oral dose in the 
morning while they are consuming the glycogen normalization diet (6.0 g carbohydrate/kg/d) for 4 days and 
once more on the carbohydrate tracer study day. Volunteers will also table a placebo (PLA) during one arm 
of the study. It will be administered in the same sequence as PIO to assure blindness to study participants 
and blinded study staff. The PLA used will be a closely matched tablet, Zeebo®, composed of 100% 
microcrystalline cellulose. PIO/PLA will be held and administered by OMSO staff only.  
 
High Altitude Exposure: To simulate ascension to high altitude, experimental trial testing will occur in the 
hypobaric/hypoxic (altitude) chamber at USARIEM under temperate (20°C, 20-30% rh) ambient conditions.  
The chamber will reduce barometric pressure, and thus ambient oxygen pressure, to a value similar to that 
at the summit of Pike Peak, CO (4,300 m). Volunteers will be exposed to hypobaric/hypoxic conditions for 
5-hrs before beginning steady-state exercise to mimic our past investigations. Total time in the chamber on 
the experimental trial day will be ~7-hrs. No more than two volunteers will be tested in the chamber at one 
time. Study personnel who will conduct data collection in the altitude chamber will be medically cleared by 
OMSO. Medical staff (OMSO) will be onsite while volunteers and staff are in the simulated altitude 
chamber. Additionally there will be at least three chamber operators in communication with the PI who will 
be in the chamber with volunteers at all times.  
 
Steady-State Treadmill Exercise: Results from a V̇ O2peak assessment under HA conditions will be used 
to prescribe intensities for treadmill exercise on experimental trial days utilizing ACSM equations to 
estimate the speed and grade of the treadmill. Volunteers will complete 80-min of metabolically-matched, 
steady-state exercise at 55  ±5% of their V̇ O2peak determined at HA. A practice session will be conducted 
during the baseline period to confirm prescribed speed and grade are appropriate to induce target V̇ O2 and 
allow the research team to make modifications to prescribed speed and grade if needed to induce target 
VO2. The practice session will range ~40-80 minutes in length. The speed and grade determined during the 
practice session will be used to induce the same absolute intensity between study arms. The mode, 
duration, and intensity of steady-state exercise were chosen to mimic our past investigations (3, 4).  
 
Carbohydrate Tracer Studies: After a 10 hr overnight fast, two catheters will be placed into the lower arm 
(one in each arm). One arm will be used for infusion of 6,6-[2H2] glucose tracer and the other will be used 
for blood sampling. Following an initial blood and breath sample collection to determine background 
enrichments, a primed, continuous infusion of 6,6-[2H2] glucose (Cambridge Isotope Laboratory, Andover, 
MA, USA) will begin (prime, 82.2 µmol∙kg-1; continuous rate, 0.78 µmol∙kg-1∙min-1) (24, 25) for 2 hrs before 
and during exercise. Volunteers will complete 80-min of steady-state exercise on a treadmill, with V̇ O2, 
V̇ CO2, HR, and SaO2 measured at approximately 5, 20, 40, 50, 60, and 75 min. During exercise volunteers 
will consume a glucose beverage at 1.8 g/min. Drinks will contain 145 g glucose, with a total volume of 
1450 mL. The drink will be consumed in four boluses throughout the 80-min of steady-state exercise (0, 20, 
40, and 60 min). The first bolus will be 550 mL, with the remaining three being 300 mL. The glucose 
beverage consumed during exercise will be enriched with 200 mg 13C-glucose (Cambridge Isotope 
Laboratory) to increase the isotopic enrichment well above natural levels and optimize the measurement of 
exogenous glucose oxidation.  
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 12 of 26 
Figure 3: Carbohydrate Tracer Studies 
 
 
Blood Sampling (credentialed procedure): Blood samples will be collected using an indwelling catheter 
kept patent using IV saline at approximately at approximately -300, -120, -30, 0, 20, 40, 60, 80, and 140 
min during exercise by a USARIEM credentialed phlebotomist. Baseline blood sampling will occur after a 
10-hr overnight fast on experimental trial days. A total of 18 blood samples will be completed during this 
study taking ~340 mL of blood sampled from each volunteer over the experimental trial days (Table 3). 
Blood samples will be used for isotope analysis assessment, substrate and hormone responses, and iron 
markers (Table 3).   
 
For glucose kinetic analysis, the tracer/tracee ratio (6,6-[2H2] glucose/glucose) using gas-chromatography-
mass spectrometry (GCMS; Metabolic Solutions, Inc., Nashua, NH) (24), while 13C/12C in plasma glucose 
will be measured using isotope-ratio mass spectroscopy (IRMS; Metabolic Solutions).  
 
Breath Sampling (non-credentialed procedure):Breath samples will be collected at approximately -300, 0, 
20, 40, 45, 50, 55, 60, 65, 70, 75, and 80 min during exercise to determine exogenous glucose oxidation 
using single-patient breath collection bags (Quin-Tron Instrument Company, Milwaukee, WI, USA). 
Baseline breath sample will also be collected prior to correct for background of naturally occurring 13C. The 
natural enrichment of the glucose ingested and the 13C/12C in expired gas samples will be analyzed using 
isotope-ratio mass spectroscopy (Metabolic Solutions, Inc., Nashua, NH).   
 
      B5.4  Data Collection   
 
Table 2.Outcome Variables 
Data Element/Variable 
Source 
Operational Specification 
Anthropometric data 
Direct measurement 
Height, weight, body composition 
Substrate oxidation 
Blood and breath  
Glucose turnover, carbohydrate, 
fat, and protein oxidation 
Metabolic response 
Blood  
Substrate analytes, hormones, 
inflammation, iron status 
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 13 of 26 
 
Table 3. Blood Analytes 
 
Time points (min) 
Analyte 
-300 
-120 
-30 
0 
20 
40 
60 
80 
1404 
Glucose1 
x 
x 
x 
X 
x 
x 
x 
X 
 
Insulin2 
x 
 
 
X 
x 
x 
x 
X 
 
Free fatty acids2 
x 
 
 
X 
x 
x 
x 
X 
 
Lactate2 
x 
 
 
X 
x 
x 
x 
X 
 
Glycerol2 
x 
 
 
X 
x 
x 
x 
X 
 
Norepinephrine2 
x 
 
 
X 
x 
x 
x 
X 
 
Epinephrine2 
x 
 
 
X 
x 
x 
x 
X 
 
Hemoglobin1 
x 
 
 
X 
 
 
 
x 
 
Hematocrit1 
x 
 
 
X 
 
 
 
x 
 
13C-glucose3 
 
 
 
X 
x 
x 
x 
x 
 
6,6-[2H2] glucose3 
 
x 
x 
X 
x 
x 
x 
x 
 
PBMC1 
x 
 
 
X 
 
 
 
x 
x 
TNFα1 
x 
 
 
X 
 
 
 
x 
x 
IL-61 
x 
 
 
X 
 
 
 
x 
x 
IL-101 
x 
 
 
X 
 
 
 
x 
x 
Hemooxygenase 11 
x 
 
 
X 
 
 
 
x 
x 
Adiponectin1 
x 
 
 
X 
 
 
 
x 
x 
Erythropoietin1 
x 
 
 
X 
 
 
 
 
 
Hepcidin1 
x 
 
 
X 
 
 
 
 
 
Soluble transferrin receptor1 
x 
 
 
X 
 
 
 
 
 
Erythroferrone1 
x 
 
 
X 
 
 
 
 
 
Platelet derived growth 
factor-BB1 
x 
 
 
X 
 
 
 
 
 
Ferritin1 
x 
 
 
X 
 
 
 
 
 
Serum Iron1 
x 
 
 
X 
 
 
 
 
 
Transferrin Saturation1 
x 
 
 
X 
 
 
 
 
 
C-reactive protein1 
x 
 
 
X 
 
 
 
 
 
Archive 
x 
x 
x 
X 
x 
x 
x 
x 
x 
 
Samples will be analyzed at 1USARIEM, 2Pennington Biomedical Research Center (Baton Rouge, LA), and 
3Metabolic Solutions (Nashua, NH). 4Final blood draw at ~140 min will be taken under normoxic conditions 
in both trials. All blood samples will be separated into plasma and serum through centrifugation, aliquoted, 
and stored at –80˚C until analysis or shipment. Study samples will be collected and aliquoted at USARIEM 
and specific samples will be shipped on dry ice to Pennington Biomedical Research Center, and Metabolic 
Solutions. Serum iron and transferrin saturation will be measured using an automated clinical chemistry 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 14 of 26 
and immunoassay analyzer (Dimension: Siemens Healthcare Diagnostic, Deerfield, IL, USA). Serum 
soluble transferrin receptor, interleukin-6, hepcidin, platelet-derived growth factor-BB, erythropoietin, 
erythroferrone, and C-reactive protein will be determined using enzyme-linked immunosorbent assays. 
Blood sent out to other laboratories for analysis will not have any remaining samples after analysis is 
completed. USARIEM will retain all archive samples. 
 
Plasma Glucose Turnover Calculations 
For calculation of plasma glucose turnover the Steele equation with modifications for non-steady state will 
be used (26). Enrichment (E) will be expressed as mole percent excess (MPE); calculated as (TTR)/(1 + 
TTR), where TTR is the tracer to tracee ratio. Appropriate corrections for skewed abundance distribution 
and overlapping spectra will be made for the TTR of the glucose tracers, 6,6-[2H2] glucose and U-13C-
glucose (26). From these calculations, total glucose Ra will be comprised of rates of appearance of 
exogenous (i.e., ingested) glucose and of endogenous (i.e., hepatic glucose production and negligible renal 
glucose production or splanchnic glucose) glucose (26): 
 
Total glucose Ra (Total Ra) = (F – ((pV x ((C2 + C1)) / 2) x ((E2 - E1) / (t2 - t1)))) / ((E2 + E1) / 2) 
Glucose Rd = Total Ra – (pV (C2 - C1) / (t2 - t1)) 
Exogenous glucose Ra (Exo Ra) = ((Total Ra + F ) x ( ( G2 + GE1) / 2 ) + ( (pV x ( (C2 + C1) / 2) ) x ((G2 - 
G1) / (t2 - t1))) 
Endogenous glucose Ra = Total Ra - Exo Ra 
Metabolic Clearance Rate (MCR) = Glucose Rd / ((C2 + C1) / 2)   
 
Where F represents the infusion rate of 6,6-[2H2] glucose; pV is the effective volume of distribution for 
glucose, C1 and C2 are plasma glucose concentrations at t1 and t2, respectively, E1 and E2 are plasma 
enrichments of 6,6-[2H2] glucose at t1 and t2, respectively, and Ed and Ep are tracer enrichments of U-13C-
glucose from the test drink and plasma, respectively.  
 
Calculations of Carbohydrate and Fat Oxidation 
Carbohydrate and fat oxidation rates will be calculated from V̇ O2 (L/min) and V̇ CO2 (L/min) during the 80-
min exercise bout as described by Jeukendrup and Wallis (27): 
 
Fat oxidation (g/min) = (1.695 x V̇ O2) – (1.701 x V̇ CO2)  
Carbohydrate oxidation (g glucose/min) = (4.585 x V̇ CO2) – (3.226 x V̇ O2)  
 
Calculations of Exogenous and Endogenous Glucose Oxidation 
Exogenous and plasma glucose oxidation will be calculated as (7): 
 
Exogenous glucose (g/min) = V̇ CO2 [(Rexp - Rref)/(Rexo - Rref)]/k 
Plasma glucose (g/min) = V̇ CO2 [(Rexp - Rref)/(Rglu - Rref)]/k 
 
where V̇ CO2 is in L/min, Rexp is the observed isotopic composition of expired CO2, Rref is the isotopic 
composition of expired CO2 at rest before ingestion of the first dose of 13C-glucose, Rexo is the isotopic 
composition of the exogenous glucose ingested, Rglu is the isotopic composition of plasma glucose, and k 
(0.747 L/g) is the volume of CO2 provided by the complete oxidation of glucose. Total endogenous glucose 
oxidation can be calculated by subtracting exogenous glucose oxidation from total CHO oxidation.  
Endogenous glucose oxidation derived from muscle and liver can be determined by subtracting plasma 
glucose oxidation from total carbohydrate oxidation (muscle), and subtracting exogenous carbohydrate 
oxidation (liver) from plasma glucose oxidation (7).  The first 40 min of steady-state exercise will allow for 
equilibration between the 13C/12C in expired CO2 and the 13C/12C in CO2 produced in tissues (28). Thus, 
endogenous glucose oxidation will only be calculated from samples obtained in the last 40 min of steady-
state exercise (40 to 80 min). 
 
Blood Analytes  
Serum glucose, free-fatty acids, glycerol, and plasma lactate concentrations will be determined using 
enzymatic and colorimetric assays (Beckman Coulter DXC 600 Pro, Beckman Coulter, Brea, CA, USA). 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 15 of 26 
Serum insulin concentrations will be determined using an advanced automated immunoassay instrument 
(ImmuliteR 2000: Siemens Healthcare Diagnostic, Deerfield, IL, USA). Serum epinephrine and 
norepinephrine will be determined using a BI-CAT® Adrenaline and Noradrenaline ELISA assay kit (Eagle 
Biosciences, Nashua, NH, USA). Blood analytes will be analyzed at Pennington Biomedical Research 
Center (PBRC) under an existing fee for service contract. 
 
Circulatory and Intracellular Inflammatory Biomarkers  
Following ~20 minutes of seated postural control, venous blood (8-10 mls) will be collected at four time 
points (-300, 0, 80, and 140 min) into EDTA treated collection tube. PBMC will be isolated using density 
gradation separation. Briefly, whole blood will be carefully layered on-top of a separating media 
(Histopaque 1077, Sigma-aldrich) at a 1:1 ratio then centrifuged at 450 x g for 30 minutes at 22°C. Blood 
plasma will be aliquoted into cyrovials and stored at -80°C freezer for batch analysis at a later time. The 
buffy coat containing the PBMC will be collected and transferred into a sterile conical tube and washed 
thrice with 10 ml phosphate buffer saline. The PBMC pellet will then be stored at -80°C for analysis of 
intracellular proteins using immunoblotting. Circulating (in blood plasma) and intracellular (PBMC) 
biomarkers will be assayed via molecular assay techniques (e.g., Western blot, enzymatic/colorimetric, 
multiplex, etc.) at the USARIEM laboratories. Intracellular inflammatory biomarkers will include, but not be 
limited to AMPK, SIRT1, NF-KB, IKK-B, and IL1RA.       
  
B5.5  Managing Data and/or Human Biological Specimens for this Research  
All data and medical information obtained will be considered privileged and held in confidence. Study 
volunteers will be assigned unique subject identification (ID) numbers that will not contain any personal 
identifiers such as name, social security number, address, date of birth, zip code, etc. This study subject ID 
number will be used on all data collection instruments, to include questionnaires, data collection forms, 
computer records, etc. A number will be assigned as each volunteer is medically cleared for participation.  
A master list linking the volunteers’ names and ID numbers will be kept in a separate locked file in the 
principal investigator's office, or kept in a computer file with password-protected access restricted to the 
principal investigator and study coordinator. When the results of the research are published or discussed in 
conferences, no information will be included that would reveal identity. All samples will be stored in -80ºC 
freezer at USARIEM in room 322 or 304 using the subject identification number. De-identified samples for 
isotopic analysis will be shipped on dry ice to (blood) and room temperature (breath) to Metabolic 
Solutions. De-identified blood will be shipped on dry ice to Pennington Biomedical Research Center. All 
samples will be shipped via FedEx and stored in these laboratories until analyzed. Once samples have 
been analyzed, there will be no remaining sample for storage other than the archived sample stored at 
USARIEM. Coded data will be transmitted between the above mentioned laboratories via an encrypted 
email, a secure file transfer site, or using an approved removable media. Only USARIEM will maintain 
digital copies of these data. Body composition and V̇ O2peak data will be shared with volunteers. No other 
data will be shared with volunteers.    
 
Only the principal investigator and study coordinator will have access to personal identifiable data. No 
outside laboratory will have access to identifiable data. Hard copy data records will be stored for a 
minimum of three years from the time the study is completed. Electronic data records will be maintained for 
a period of at least ten years after the study has been completed. The master list will be destroyed upon 
the protocol’s closure. 
 
      B5.6  Managing Data and/or Human Biological Specimens for Future Research   
 
De-identified study samples will be stored in -80ºC freezer at USARIEM in room 322 or 304 for potential 
future use and maintained indefinitely. Only personnel assigned to the research study by the principal 
investigator will have access to samples. The de-identified data and samples will remain under the control 
of the PI and may be shared with outside collaborators for future research. Any use of the samples outside 
of this defined protocol will be submitted as a protocol amendment or a new protocol.   
   
      B5.7  Devices, Drugs, Dietary Supplements, Nutritional Supplements, And Biologics   
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 16 of 26 
                B5.7.1  Devices 
 
5.7.1.1 FDA-approved device being used in this research according to the approved labeling 
DEXA, DPX-IQ, Lunar Corporation, True Max 2400, Parvomedics, Sandy, Utah, USA, Polar Electro 
Inc, Oulu, Finland 
 
5.7.1.2 FDA-approved device being used in this research in a manner other than its approved 
labeling  
N/A 
 
5.7.1.3 Any device not approved by the FDA 
      N/A 
 
                B5.7.2  Drugs 
 
B5.7.2.1 FDA-approved and used in accordance with the approved labeling 
      
B5.7.2.2 FDA-approved and used in a manner not in accordance with its approved labeling 
Pioglitazone, Thiazolidinedione, Takeda Pharmaceutical Company 
 
  
B5.7.2.3 Any drug not approved by the FDA 
N/A 
      
B8  Statistical Analysis 
 
B5.8.1  Sample Size Estimation 
Based on results of exogenous carbohydrate oxidation and MCR rate between SL and HA in our previous 
investigation (3, 4), a sample size of 8 is required to achieve 80% power (Table 4). We will request to 
enroll up to 24 individuals to account for withdrawals from the study. 
 
 Table 4: Previous study effect sizes 
Study 
Outcome 
Delta 
Effect Size 
Sample Size 
Young et al. (4) 
Exogenous Glucose 
Oxidation 
0.35 ± 0.21 
1.11 
7 
Margolis et al. (3)   
Exogenous Glucose 
Oxidation 
0.09 ± 0.06 
0.95 
8 
Margolis et al. (3)  
MCR 
3.1 ± 2.0 
0.98 
7 
 
B5.8.2  Data analysis 
Statistical analyses will be conducted using either SPSS (IBM Corp. Armonk, NY), SAS 9.3 (SAS Institute 
Inc., Carey, NC), or equivalent. Shapiro Wilks will be used to assess all data for normality. Data that are not 
normally distributed will be log transformed for statistical analysis. Paired T tests will be used to assess 
effects of study arms (HA+PIO vs. HA+PLA) on chamber conditions (barometric pressure, temperature, 
and humidity), oxygen saturation, heart rate, V̇ O2, V̇ CO2, respiratory exchange ratio, ventilation exchange, 
total glucose and fat oxidation, plasma glucose, muscle, and liver oxidation. Mixed Model repeated 
measures ANOVA will be used to assess the effects of condition, time, and their interactions for breath 13C-
enrichments, exogenous glucose oxidation, and blood analytes. Bonferroni adjustments for multiple 
comparisons will be performed if significant interactions are observed.  
 
 
 
SECTION C:  HUMAN RESEARCH PROTECTIONS 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 17 of 26 
 
C1.  RECRUITMENT AND CONSENT   
 
       C1.1  Identification and Selection of Subjects   
Volunteers will be recruited from the federally and non-federally employed civilian population, the Natick 
Human Research Volunteer (HRV) Pool, NSSC active duty military personnel, and the active duty 
population located at other military organizations, to include, but not limited to coordination with the NSSC 
Soldier and Squad Optimization and Integration Team (SSO-IT). 
 
Civilian volunteers and other active duty personnel will be recruited by “word of mouth”, posted flyers, or 
electronic distribution of the “off-site” flyer to include a text-only, approved version of the flyer. Recruiting 
materials will be distributed around NSSC, surrounding community and organizations, and on bulletin 
boards (physical and virtual/electronic) at surrounding universities or other organizations. The text-based 
flyer will be posted on various USARIEM social media sites and may be used in distribution media requiring 
a text format (e.g., electronic newsletters). Recruiting may also be conducted through information meetings 
presented to college classes, clubs, sports teams, or other organizations. Approvals from the requisite 
parties will be obtained prior to any recruitment activities.  
 
      C1.2  Recruitment Process  
Consent briefings will be conducted either in-person or virtually using a video communication platform (i.e., 
Microsoft Teams, Zoom, Facetime, or similar). 
 
For Soldiers in the U.S. Army Natick Soldier System Center Human Research Volunteer (HRV) Program, 
the Principal Investigator will furnish a copy of the consent form to the Human Research Volunteer 
Program Coordinator or designee. The HRV Coordinator will schedule the consent briefing for the HRV 
platoon.  
 
Volunteers from other military organizations (i.e., non-HRV military) may be recruited through coordination 
with NSSC SSOIT or similar avenues. Non-HRV military may be recruited around NSSC. The NSSC SSO-
IT Coordinator, or respective Unit Leadership, may schedule the consent briefing for the non-HRV military. 
Non-HRV military coming to Natick to participate in research on temporary duty station (TDY) permissions, 
will receive transportation and lodging paid for by the study. Additionally, compensation for meals will be 
provided at the local per diem rate for days that research volunteers are not following the controlled study 
diet.  
 
An ombudsperson will be present during all group briefings for military personnel.  
 
Superiors of Service members (e.g., unit officers, senior NCOs, and equivalent civilians) / supervisors of 
DoD civilians (e.g., military and civilian supervisors or anyone in the supervisory structure) shall not be 
present at any recruitment sessions or during the consent process in which members of units under their 
command / personnel under their supervisions are afforded the opportunity to participate as human 
subjects of research. 
 
For civilian volunteers, the Principal Investigator will provide a copy of the informed consent document to 
the individual. The Study Coordinator will schedule the consent briefing for the civilian volunteers. An 
ombudsperson will not be present during either individual or group briefings.  
     
       C1.3  Eligibility   
Following the completion of written consent, volunteers will also undergo the OMSO medical clearance 
process for participation in accordance with USARIEM procedures outlined for screening volunteers for 
research involving exercise.  This includes completing a two part medical clearance: 1) general medical 
clearance to confirm general health status and 2) specific medical clearance to confirm specific eligibility for 
the current study, to include an ECG. Screenings will be conducted and reviewed by a licensed physician 
or physician assistant from OSMO. OMSO will travel to the site of study recruitment to conduct study-
specific medical clearances. Final eligibility will be determined by the Principal Investigator using 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 18 of 26 
information from the OMSO medical clearances. All volunteers will undergo the required clearances in 
order to participate. Volunteers recruited through NSSC SSO-IT, or equivalent, may undergo medical 
clearance at their home duty station prior to arrival at USARIEM (clearances will be coordinated between 
the Principal Investigator, OMSO, and the unit’s Brigade Surgeon or similar).  
 
Volunteers will be screened for anemia. Final classification of anemia, and therefore eligibility to participate, 
will be determined by OMSO.  
 
All volunteers must be willing to consume only the food and beverages provided by study staff during the 
controlled feeding portions of this research study, and they must be willing to adhere to exercise and 
physical activity prescriptions and restrictions. 
 
If the results of all screening tools reveal the volunteer fits the screening criteria, they will be eligible to 
participate in the study.   
 
       C1.4  Consent Process   
Prior to providing informed consent, discussions with potential volunteers (such as over the telephone) will 
involve answering general questions, but will not involve formal screening nor the collection of any 
personally identifiable information besides their name, email, and telephone number. No study procedures 
will occur prior to any volunteer giving their informed consent.  
 
The Principal Investigator, an Associate Investigator or the Study Coordinator will brief potential volunteers 
about the nature, purpose, procedures involved, risks, expectations and requirements for participation in 
the study. Prospective volunteers will be familiarized with the study procedures and informed verbally and 
in writing of their rights to withdraw from any part of the study without penalty or prejudice. The Principal 
Investigator or designee will answer all group and private questions. Potential volunteers will have at least 
one hour after they are briefed to read and review the Informed Consent document. Potential volunteers 
who are ready to provide their written, informed consent may be encouraged to do so after the briefing. 
Potential volunteers who would like to take more time to consider whether they wish to participate in the 
research will be given the option of returning the consent form at a later time (i.e., by early the next morning 
or in the subsequent days). 
 
 Documentation of informed consent may be captured in the form of an electronic signature (i.e. common 
access card signatures for military or federal personnel). A copy of the informed consent document will be 
provided to the volunteer with the original kept for study documentation. Volunteers who have already 
consented will be informed of any new information or changes to the protocol that may affect their 
willingness and ability to continue participation in the study using an approved consent addendum.  
 
In the case of COVID risk mitigation plans, briefings can be done virtually and consent forms can be 
emailed and digitally signed. 
 
 
C1.4.1  Research involving subjects with cognitive impairment or who lack capacity to 
provide informed consent 
 
     N/A 
 
                 C1.4.2  Research involving non-English speaking subjects 
 
     N/A 
 
 
C1.4.3  Research involving a waiver of the requirement to obtain informed consent OR 
alteration of the elements of informed consent 
 
     N/A 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
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C1.4.4  Research involving a waiver of the requirement for investigator to obtain a signed 
consent form  
 
     N/A 
 
                 C1.4.5  Waivers of assent or parental permission when the research involves children 
 
     N/A 
 
                 C1.4.6  Research involving data collection for the USAMRDC Volunteer Registry Database 
 
 
N/A 
 
C2.  COMPENSATION FOR PARTICIPATION   
 
Volunteers who participate in the study will receive $50.00 per blood sample (9 samples per CHO tracer trial 
day; 2 CHO tracer trial days per volunteer equals 18 samples for entire study), for a total of $900.00 for 
completing the study. Volunteers will not be compensated for blood draws completed during the 
screening/medical clearance visit. Should volunteers withdraw, they will be compensated for any blood 
samples they complete except for the initial blood sample during the medical screening. Payments will 
processed when the volunteer concludes the study (end of study period or voluntary withdrawal), as a total 
lump-sum payment, received as a direct deposit.   
 
Note: Participants who receive more than $600 in a calendar year will have this income reported to the Internal 
Revenue Service. 
      
C3.  WITHDRAWAL FROM RESEARCH PARTICIPATION   
 
Volunteers may withdraw from the study at any time without penalty or loss of benefits to which they would 
otherwise be entitled. They will do so by informing the Principal Investigator, Associate Investigator, or a staff 
member of their intent to withdraw verbally or in writing (electronic or paper/pencil). They will then be asked to 
verbally discuss their choice to withdraw with the Principal Investigator so that reasons for withdrawal can be 
documented. However, volunteers will not be required to discuss why they withdrew, as the volunteer may 
choose to discontinue participation without providing reason. An Investigator may stop an individual’s 
participation in the study if the volunteer is unwilling or unable to complete study procedures. If an individual 
needs or wants to withdraw when in the altitude chamber, the volunteer will descend to SL in a separate part of 
the chamber with a study staff member, and be released from the altitude chamber at the time of withdrawal. 
An Investigator may also withdraw a volunteer if the individual becomes ill or injured or it would not be in the 
volunteer’s best interest to continue. If the participant is withdrawn by the Investigator or decides to voluntarily 
withdraw him/herself, all further data collection will discontinue. Any de-identified data and biospecimens 
collected up until the point of withdrawal will be retained for future use and stored with all other study data. 
Volunteers will be compensated for any blood samples they completed up until that point, and they will be 
asked to return any remaining food items that were provided, in addition to any wrappers and diet/activity logs 
that they had completed up to the point of withdrawal. 
     
C4.  PRIVACY FOR SUBJECTS   
 
To protect the volunteer’s privacy, all of their research-related records will be labeled or “coded” with an 
assigned research volunteer number that will not include their name or any other form of identifiable 
information.  The principal investigator or study coordinator will keep the link between volunteer number and 
the volunteer’s research records in a locked cabinet.  Any documents that will require the volunteer’s name, 
such as the consent form, will be kept in a locked cabinet separate from any research documents that contain 
the volunteer’s ID number.  The principal investigator and study coordinator are the only people who will be 
able to match the research volunteer number with any of their personal identifying information.    
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 20 of 26 
When the results of the research are published no information will be included that would reveal the volunteer’s 
identity to others. Photographs, videos, or audio-tape recordings of volunteers will only be used, if the 
volunteer grants permission through the Audio/Visual Image Release form. Each volunteer will also be asked 
to grant permission for his/her name to be included on his/her photo or video image or in writing connected to 
his/her image.  If a volunteer does not grant permission through the Audio/Visual Image Release, then no 
photos or other visual recordings will be taken of him/her. In the event that it is discovered that an individual 
has been inadvertently photographed or visually recorded without his/her permission, the materials will be 
immediately destroyed.  Permission through the Audio Visual Image Release form will be confirmed before any 
photographs or other visual recordings are used. 
      
C5.  CONFIDENTIALITY PROCEDURES FOR RESEARCH RECORDS, DATA, HUMAN BIOLOGICAL 
SPECIMENS   
 
Complete confidentiality cannot be promised to military participant because information bearing on the military 
participant health may be required to be reported to appropriate medical or command authorities.   
 
All data and medical information obtained will be considered privileged and held in confidence.  A unique study 
ID number will be assigned to each participant that will not contain any personal identifiers such as name, 
social security number, address, date of birth, zip code, etc. and that only this study participant ID number will 
be used on all data collection instruments, to include questionnaires, data collection forms, computer records, 
etc.  A number will be assigned as each volunteer is medically cleared for participation.  The master key linking 
ID numbers to individuals will be destroyed when the study is closed. When the results of the research are 
published or discussed in conferences, no information will be included that would reveal identity. 
      
C6.  RISKS OF HARM, MEASURES TO REDUCE THE RISKS OF HARM, AND BENEFITS OF 
PARTICIPATION 
 
       C6.1 Risks of Harm   
Research Procedure Name:  Dual energy X-ray absorptiometry (DEXA) scan 
Research Procedure Description: Volunteer will lay face-up on the DEXA densitometer table in 
shorts, t-shirts, and stocking feet.  Volunteers will be asked to remain motionless for the 8-10 min scan. 
Research-related Risks:  Exposure to less than 0.6 mrem Xirradiation in a slowspeed (up to 20 
minutes) wholebody scan is possible.  This dose is equivalent to approximately 1/500 of normal annual 
background radiation (300 mrem/year), 1/6 of the radiation received in a transatlantic flight (3.825 
mrem), or 1/3 of the radiation received in a chest X-ray (2 mrem). 
Measures to Minimize Risks of Harm:  A quality assurance check will be completed on the DEXA 
each day prior to its use; the software will not allow the use of the DEXA densitometer if the quality 
assurance check fails. Volunteers must be advised that the health effects of very low level exposures to 
ionizing radiation, if any, are unknown (thought to be minimal).  
 
Research Procedure Name:  Indwelling Catheters 
Research Procedure Description:  A needle will be used to guide a catheter into the antecubital vein 
of the volunteer. The catheter will be attached to saline to keep the line patent for multiple blood 
samples.     
Research-related Risks: The risks of blood sampling are small and usually limited to local bruising or 
swelling.  Also sometimes volunteers feel faint or may faint. If the volunteer has had problems with 
fainting during blood sampling in the past, they may be more prone to them during future procedures. If 
the catheter, the tube that is left in the arm after the needle is removed, becomes clogged at any time 
during the protocol, we will have to replace this to continue blood sampling. This will require another 
needle to be inserted into your arm. In addition, the catheter can cause irritation, bruising, swelling, 
infection, or an allergic reaction. 
Measures to Minimize Risks of Harm: Trained technicians will use sterile techniques to place the 
catheter; however, in spite of being careful there is a chance that the site may become infected.  
Subjects will be briefed on the signs and symptoms of potential complications and given information on 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
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who to contact for medical assessment and/or treatment. Volunteers should not donate blood for eight 
weeks before or after this study.  
 
Research Procedure Name: 6,6-[2H2] glucose infusions  
Research Procedure Description: 6,6-[2H2] glucose will be infused using a indwelling catheter. 
Research-related Risks: The primary risks associated with tracer studies are those related to venous 
catheterization. The catheter can cause irritation, bruising, or infection. There are no known risks or 
reported side effects associated with administration of 6,6-[2H2] glucose infusions to humans during 
clinical or experimental studies. The risks associated with the infusion include volume overload, 
infection, and allergic reaction to the infused substance. There have been no occurrences of volume 
overload, no occurrences of infection or allergic reaction attributable to iodine used prior to 
venipuncture in any of the 200 infusion protocols that the investigators have been involved. To minimize 
the likelihood of these events occurring, infusion rate will be closely monitored and maintained at less 
than 30 ml/hr throughout the entire infusion experimental trial day. 
Measures to Minimize Risks of Harm: All staff who directly participate in the infusion studies will be 
properly trained how to safely monitor (i.e., infusion pumps and IV lines) infusion studies from Dr. 
Margolis, who has extensive experience with infusion studies. In addition, infusates will be prepared 
sterile, pyrogen-free, and in the proper dosages by a licensed pharmacist. If sign of allergic reaction, 
rash or redness, infusion will be stopped immediately.  
 
Research Procedure Name:  Venipuncture 
Research Procedure Description: A needle will be used to obtain single blood samples of the 
antecubital vein.   
Research-related Risks:  Venipuncture is a routine clinical procedure the medical community 
commonly uses to obtain blood samples. The immediate complications may be slight pain during the 
entry of the needle into the skin, possible dizziness, and syncope. Dizziness or syncope constitutes no 
long-term harm, and immediate relief is achieved by having the subject put their head down between 
their knees or lie down. Additionally, a hematoma may result from the venipuncture, but this is more 
unsightly than risk producing. Late complications might include thrombosis of the vein due to trauma or 
infection. These complications are extremely rare. 
Measures to Minimize Risks of Harm: Participant monitoring, aseptic technique, including sterile 
disposable blood collection apparatus and adherence to standard medical precautions reduce risk.  
Trained technicians will perform all venipuncture.  
 
Research Procedure Name:  Exercise 
Research Procedure Description:  Exercise testing will occur on a cycle ergometer or treadmill.  
Exercise will be at various levels of intensity based on exercise protocol. 
Research-related Risks:  Exercise per se rarely provokes cardiovascular events in healthy individuals 
with normal cardiovascular systems.  The prevalence of fatal events in the U.S. is approximately 
1:133,000 to 1: 185,000 for men and 1:769,000 to 1:1,500,000 for women who are competitive high 
school and college athletes. For middle-aged and older adults the relative risk rises to 1:15,000 to 
1:18,000 for individuals without a prior history of cardiovascular events. Current civilian and military 
guidelines state that individuals less than 40 years of age who have no symptoms of or known 
presence of heart disease or major coronary risk factors have a low risk for cardiac complications 
during vigorous exercise.  All volunteers in this study fall into this low risk category.  Local muscle 
discomfort and fatigue may occur in active muscles during and shortly after exercise.  Muscle soreness, 
ranging in intensity from mild to severe, may persist for 1 to 7 days.  
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Studies have confirmed the safety 
of maximal exercise testing, particularly among apparently healthy persons without significant 
cardiovascular risk factors.  As a precaution, there will be at least one spotter during all exercise 
sessions, and heart rate will be monitored in real time during testing.  In addition, exercise monitors and 
test administrators will be CPR-certified.  
 
Research Procedure Name: Oral Pioglitazone administration        

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 22 of 26 
Research Procedure Description: Participants will be administered 15 mg of PIO, an insulin 
sensitizing agent, for five days during one arm of the research trial. Study staff will observe research 
volunteers when PIO is administered.  
Research-related Risks:  The most common side effects of PIO include cold-like symptoms (upper 
respiratory tract infection), headache, sinus infection, muscle pain, and sore throat. Rare but possible 
side effects can include hypoglycemia, fluid retention which leads to swelling (edema), and weight gain. 
Chronic use of PIO has been associated with increased risk of bladder cancer. 
Measures to Minimize Risks of Harm: (Precautions, safeguards): To minimize side effects the 
study will use a low dose of PIO, 15 mg, which is considered a starting dose when medication is initially 
prescribed. PIO will be administered for short-term use (5 days), which is significantly less dose 
exposure time for side effects, and particularly more rare side effects to occur. Additionally, PIO will be 
administered as a monotherapy, without presence of other hypoglycemia agents such as insulin, and 
diet and exercise will be controlled which will nearly eliminate risk of hypoglycemia. The study will 
recruit healthy, male volunteers who will be medically screened to eliminate volunteers with known 
comorbidities which could exaggerate side effects. OMSO will be responsible for administering PIO to 
volunteers and medical oversight of volunteers.          
 
Research Procedure Name:  Altitude Exposure 
Research Procedure Description: Participants will exercise at 4300 m simulated altitude.    
Research-related Risks:  Hypoxemia, lightheadedness, AMS, HAPE, HACE, ear pain/discomfort, and 
peripheral/facial edema. 
Measures to Minimize Risks of Harm: (Precautions, safeguards):   
• 
During hypobaric exposures, volunteers will always be accompanied by one or more trained staff 
that will monitor signs and symptoms of research-related risks. To additionally minimize risk, 
volunteers will be familiarized with safety procedures and equipment in the hypobaric chamber. 
Additionally, medical staff (OMSO) will be onsite while volunteers and staff are in the simulated 
altitude chamber.  
• 
Hypoxemia- Exposure to hypobaric hypoxia introduces the hazard of hypoxemia.  Depending upon 
the degree and duration of hypobaric exposure, hypoxemia may be well tolerated without 
consequences, or may present a full spectrum of clinical entities ranging from mild discomfort to 
potentially lethal conditions. Participants will be monitored by study staff and participants will be 
allowed to terminate testing early if they desire. 
• 
Lightheadedness- Exercising at altitude can cause an increased risk of lightheadedness which 
may occur just after stopping heavy exercise. Lightheadedness increases the risk that the 
volunteer could fall. This risk (rare) will be minimized by having at least one staff member in close 
proximity to the volunteer (as is done for all exercise tests regardless of location) so that he/she 
will not fall off.  If a volunteer becomes lightheaded and feels faint, he/she will be assisted off the 
treadmill and instructed to lay down with feet up until fully recovered. Volunteers will be assisted on 
and off exercise and other experimental apparatus as needed.  
• 
AMS- AMS is a self-limited syndrome that is common when unacclimatized lowlanders are 
exposed to higher altitudes. Symptoms of AMS include headache, nausea, anorexia, lethargy, 
dizziness, tiredness, weakness, insomnia, and sometimes vomiting. The prevalence and severity 
of AMS increase directly in proportion to ascent rate and elevation. With rapid ascent, and no 
beneficial treatment (e.g., breathing supplemental O2) symptoms commonly appear within four to 
six hours. Symptoms of AMS will reach their severity within 18 to 24 hrs of altitude exposure, and 
be the most severe after awakening. With rapid ascent to the altitudes of 3000 m and 4000 m for a 
24-hr period, more than 30-90% of the untreated volunteers will likely experience AMS, with 
symptoms ranging from mild (10 to 40%), to moderate (20 to 40%) to severe (10 to 20%). Due to 
the duration (~7-8 hrs) of the altitude exposure in the current study, AMS may occur but should not 
reach its most severe symptoms.  
• 
HAPE/ HACE- The incidence of clinical HAPE in unacclimatized sea-level residents being exposed 
to high altitude for at least a few days appears to be less than 1%. As a clinical condition, the 
possibility of HACE occurring when sea-level residents are exposed to high altitude appears to be 
even lower than the incidence of HAPE. If either or both are recognized early and treated by 
descent, they are completely reversible. With regard to the proposed study, the risk of any 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
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volunteer developing either HAPE or HACE is considered remote and is not expected. If a 
volunteer shows developing symptoms of HAPE or HACE they will be removed from the simulated 
high altitude environment.  
• 
Ear Pain/Discomfort- Changes in ambient gas pressure during induction of or return from 
hypobaric conditions can cause untoward effects due to gas trapped in the body.  At the relatively 
slow rates of pressure change during normal operation (simulated ascent and/or descent) of the 
hypobaric chamber, some individuals may experience discomfort in their ears, paranasal sinuses, 
teeth, or abdomen.  In healthy individuals, pressure in the middle ear and sinuses can be 
equalized with ambient atmospheric pressure by swallowing, yawning, or tensing the muscles of 
the throat, procedures that contract the pharyngeal muscles and open the Eustachian tubes, 
thereby ventilating the middle ear and sinus.  Another more effective means of ventilating the 
middle ear and sinuses is forced expiration against a closed nose and mouth.  Adjunctive therapy 
may also include the use of short-acting antihistamines or decongestants to reduce swollen 
Eustachian tubes and sinus openings due to inflammation or infection.  During recompression 
(descent), moderately severe ear pain may be experienced by volunteers who cannot maintain 
Eustachian tube patency.  If ear or sinus pain occurs, recompression will be stopped and the 
affected volunteer will be decompressed until the pain resolves; recompression will then be 
restarted at a slower rate.  Damage to eardrums is likely only under the unusual situation where 
deliberately rapid recompression is necessitated by dire emergency.  
• 
Peripheral/Facial Edema- Peripheral and facial edema occurs in some individuals when initially 
exposed to high altitude. It is characterized by pronounced edema of the face and upper 
extremities, decreased urine output and weight gain. Although uncomfortable, it is a benign 
condition. The peripheral and facial edema resolves with descent. 
 
      C6.2  Incidental or Unexpected Findings   
All incidental or unexpected health findings identified by OMSO during the screening process or throughout 
the research process will be documented and disclosed to the volunteer. The volunteer will be encouraged 
to make an appointment with their primary care provider for a full evaluation of any identified problems. 
Volunteers with evidence of any physical, mental, and/or medical conditions that would make the proposed 
studies relatively more hazardous will be excluded.  
 
Any results perceived to be clinically relevant will not be shared with participants, as biological samples will 
not be analyzed until after participants have finished data collection. 
 
Body composition, exercise performance measures, and baseline dietary intake data may be shared upon 
request at the individual level once the requesting individual completes all study procedures.  
    
      C6.3  Potential Benefits   
 
There are no direct health or other benefits related to participation in this study. Information gathered from 
this research may benefit society in the future.   
 
C7.  DATA AND SAFETY MONITORING   
The Principal Investigator will be responsible for monitoring the data collected. The Principal Investigator, with 
the assistance of the Study Coordinator, will ensure that the data is being collected according to the methods 
described in the protocol. Electronically collected data will be downloaded daily and checked for quality. This 
also includes continuous evaluation of the following: recruitment, the informed consent process, adverse 
events, protocol adherence, and protocol deviations. This will occur continuously in order to identify 
unanticipated problems or risks to the volunteers associated with the research. The Principal Investigator will 
ensure that the number of volunteers recruited for this study complies with the protocol. If adverse events 
occur, the Principal Investigator will submit a monthly summary of the related adverse events to OMSO to 
determine whether the number of adverse events is excessive for the risks outlined in the research protocol. 
The Principal Investigator and Study Coordinator will be responsible for ensuring that the appropriate 
regulatory and IRB documentation is on file and up to date. 
 

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 24 of 26 
C8.  REPORTABLE EVENTS    
 
       C8.1  Expected adverse events  
 
An adverse event is defined as any untoward or unfavorable medical occurrence in a human research 
participant, including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or 
disease, temporally associated with the individual’s participation in the research, whether or not considered 
related to the individual’s participation in the research. 
 
Expected adverse events which are not serious are reported to the IRB at the time of continuing review of 
the protocol. These events include some level of AMS under HA conditions, local bruising and soreness 
from blood sampling, and muscle soreness or pain from exercise. 
 
All medical events that the USARIEM Office of Medical Support and Oversight (OMSO) evaluates will be 
reported to the ORQC. 
      
       C8.2  Unexpected adverse events and unanticipated problems 
 
A serious adverse event is any adverse event temporally associated with the subject’s participation in 
research that is fatal, life-threatening, permanently disabling, requires inpatient hospitalization, or results in 
congenital anomalies/birth defect, overdose or cancer, or based on appropriate medical judgment, may 
jeopardize the participant, or may require medical or surgical intervention to prevent one of the above 
outcomes. 
 
 
All medical events will be reported to USARIEM’s Office of Medical Support and Oversight (OMSO). OMSO 
staff will retain a copy of the report in the subject’s OMSO medical file as a means of tracking and 
analyzing trends in medical events.  
 
All unanticipated problems involving risk to subjects or others, serious adverse events that are unexpected 
and determined to be at least possibly or definitely related to study participation, will be promptly reported 
within one working day by phone (508-206-2371/2200) or email (usarmy.natick.medcom-
usariem.mbx.usariem-rqc-protocol@health.mil) to the USARIEM ORQC and the Commander. These 
events will also be reported to the HQ USAMRDC IRB within one working day by phone (301-619-6240), or 
by e-mail (usarmy.detrick.medcom-usamrdc.other.irb-office@mail.mil)  
 
Adverse events assessed by the PI as not serious and serious adverse events that are deemed to be 
unrelated to participation in the study will be reported to the IRB at the time of continuing review of the 
protocol.  
 
In the event of a medical emergency at facilities on the Natick Soldier Center, the local Emergency Medical 
Services (EMS) will be contacted immediately by dialing 5911. The installation security personnel will direct 
the ambulance to the proper location on the installation. While awaiting their arrival, Basic Life Support will 
be rendered by study personnel or on-site medical coverage. EMS response time to USARIEM is 
approximately 5 minutes. Transport time to definitive care is approximately 8 minutes. 
 
       C8.3  Adverse device effects 
 
     N/A 
 
       C8.4  FDA-regulated research under IND and IDE 
The investigation is not intended to be reported to the FDA as a well-controlled study in support of a 
new indication of use or any other significant labeling or advertising for the drug. The investigation will 
be conducted in compliance with the requirements for institutional review per 21 CFR Part 56 and the 
requirements for informed consent per 21 CFR Part 50. The drug will not be promoted as safe or 
effective and the study will be conducted in compliance with 21 CFR Part 312.7.  

Protocol Title:  Effects off Pioglitazone on Exogenous Carbohydrate Oxidation during Steady-State Exercise at High Altitude 
Date:  24 April 2023 
 
HQ USAMRDC IRB Research Protocol Template v_21 SEPTEMBER 2020                                                                                                                                              Page 25 of 26 
 
 
 
 
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2. 
Pasiakos SM, Karl JP, Margolis LM. Challenging traditional carbohydrate intake recommendations for optimizing 
performance at high altitude. Curr Opin Clin Nutr Metab Care 2021;24(6):483-9. doi: 
10.1097/MCO.0000000000000782. 
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Margolis LM, Wilson MA, Whitney CC, Carrigan CT, Murphy NE, Radcliffe PN, Gwin JA, Church DD, Wolfe RR, 
Ferrando AA, et al. Acute hypoxia reduces exogenous glucose oxidation, glucose turnover, and metabolic 
clearance rate during steady-state aerobic exercise. Metabolism 2020;103:154030. doi: 
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Young AJ, Berryman CE, Kenefick RW, Derosier AN, Margolis LM, Wilson MA, Carrigan CT, Murphy NE, 
Carbone JW, Rood JC, et al. Altitude Acclimatization Alleviates the Hypoxia-Induced Suppression of Exogenous 
Glucose Oxidation During Steady-State Aerobic Exercise. Front Physiol 2018;9:830. doi: 
10.3389/fphys.2018.00830. 
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Hill GW, Gillum TL, Lee BJ, Romano PA, Schall ZJ, Hamilton AM, Kuennen MR. Prolonged treadmill running in 
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Salminen A, Hyttinen JM, Kaarniranta K. AMP-activated protein kinase inhibits NF-kappaB signaling and 
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0748-0. 
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Yang H, Zhang W, Pan H, Feldser HG, Lainez E, Miller C, Leung S, Zhong Z, Zhao H, Sweitzer S, et al. SIRT1 
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Zhang WY, Schwartz EA, Permana PA, Reaven PD. Pioglitazone inhibits the expression of inflammatory 
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Coletta DK, Sriwijitkamol A, Wajcberg E, Tantiwong P, Li M, Prentki M, Madiraju M, Jenkinson CP, Cersosimo E, 
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SECTION E:  ABBREVIATIONS AND ACRONYMS 
 
U.S. Army Research Institute of Environmental Medicine; USARIEM, LC-Tandem Mass Spectrometer; LC-
MS/MS, Isotope Ratio Mass Spectrometer; IRMS, Pennington Biomedical Research Center; PBRC, College of 
American Pathologists; CAP, High Altitude; HA, Sea Level; SL, Placebo; PLA, Glucose Rate of Appearance; 
Ra, Glucose Rate of Disappearance; Rd. Metabolic Clearance Rate; MCR, Multi-Domain Operations; MDO, 
Modular Ration Enhancement Operations HA; MORE-HA, Pioglitazone; PIO, Biomedical Performance 
Enhancement; BPE, Delegation of Authority; DOA, Dual Energy X-ray Absorptiometry; DEXA, Peak Oxygen 
Uptake; VO2peak, Identification; ID, Human Research Volunteer; HRV, Solider and Squad Optimization and 
Integration Team; SSO-IT, Office of Medical Support and Oversight; OMSO, Acute Mountain Sickness; AMS, 
High Altitude Peripheral Edema; HAPE, High Altitude Cerebral Edema; HACE 
 
      
 
SECTION F: DoD PRIVACY RULE AND PROTECTED HEALTH INFORMATION (HIPAA) 
 
 
NA – will not use or disclose protected health information