Influence of Carbohydrate Availability on Skeletal Muscle and Circulating microRNA Expression
Phase: NA
Conditions: Glycogen Depletion
Sponsor: United States Army Research Institute of Environmental Medicine

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
17-17H Glycogen And Microrna Protocol_23MAY2019_V.7_CLEAN 21OCT14 
 
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PROTOCOL TITLE:  Influence of carbohydrate availability on skeletal muscle and circulating 
1 
microRNA expression 
2 
 
3 
SECTION A:  RESEARCH TEAM AND LOCATIONS 
4 
 
5 
A1.  RESEARCH TEAM 
6 
 
7 
Study Role 
Institution/Company and Contact Information 
Sponsor 
Organization/Institution/Company:  US Army Research 
Institute of Environmental Medicine (USARIEM) 
Military Nutritoin Division (MND) 
Address:  10 General Greene Ave, Bldg 42, Natick, 
MA 01760 
Point of Contact:        
     Name and Degree:  Scott J Montain, PhD 
    Title:  Division Chief 
Phone Number:  508-233-4564 
Email:  scott.j.montain.civ@mail.mil 
 
Principal Investigator 
Name, Rank, and Degree: Lee M Margolis, PhD, RD 
Title:  ORISE Post Doctoral Fellow 
Institution:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick MA 
01760 
Phone Number:  508-233-4591 
Email:  lee.m.margolis.ctr@mail.mil 
 
Associate Investigator(s) 
 
Name, Rank, and Degree:   Andrew J. Young, PhD 
Title: Nutritional Physiologist, ORSIE Fellow 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-5141 
Email:  Andrew.j.young.ctr@mail.mil 
 
Name, Rank, and Degree:  Scott J. Montain, PhD 
Title:  Division Chief 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-4564 
Email:  scott.j.montain.civ@mail.mil 
 
Name, Rank, and Degree:  James P. McClung, PhD 
Title:  Deputy Chief 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-4979 
Email:  james.p.mcclung8.civ@mail.mil 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
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Name, Rank, and Degree:  Stefan M. Pasiakos, PhD 
Title:  Nutritional Physiologist 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-6474 
Email:  stefan.m.pasiakos.civ@mail.mil 
 
Name, Rank, and Degree:  Nicholas D Barringer, MAJ, PhD, RD 
Title:  Nutritional Physiologist 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233-4599 
Email:  nicholas.d.barringer.mil@mail.mil 
 
Other Key Research  
Personnel (as applicable) 
 
Name, Rank, and Degree:  Marques Wilson, MS 
Title:  Project Coordinator 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  508-233- 5681 
Email:  marques.a.wilson.civ@mail.mil 
 
Other Individuals 
Supporting  
the Research (as 
applicable) 
 
Name, Rank, and Degree:  Claire Whitney, MS, RD 
Title:  Research Dietitian, ORISE Fellow 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-4875 
Email:  claire.c.whitney.ctr@mail.mil 
 
 
Name, Rank, and Degree:  Nancy Murphy, MS 
Title:  Laboratory Manager 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-4598 
Email:  nancy.e.murphy5.civ@mail.mil 
 
 
Name, Rank, and Degree:  Christopher Carrigan, BS 
Title:  Research Assistant, ORISE Fellow 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508) 233-5101 
Email:  christopher.t.carrigan.ctr@mail.mil 
 
Name, Rank, and Degree:  Anthony Karis, BS 
Title:  Research Physical Scientist 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-4754 
Email:  anthony.j.karis.civ@mail.mil 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
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Name, Rank, and Degree:  Nicholes J. Armstrong, MS, RD 
Title:  Project Manager 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-5153 
Email:  nicholes.j.armstrong.civ@mail.mil 
 
Name, Rank, and Degree:  Laura Lutz, MS, RD 
Title:  Dietitian 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-6492 
Email:  laura.j.lutz2.civ@mail.mil 
 
Name, Rank, and Degree:  Adrienne Hatch, MS, RD 
Title:  Dietitian 
Institution/Company:  USARIEM / Military Nutrition Division 
Address:  10 General Greene Ave, Bldg 42, Natick, MA 01760 
Phone Number:  (508)233-5648 
Email:  adrienne.m.hatch.civ@mail.mil 
 
Name, Rank, and Degree:  Rasha Hammamieh, PhD 
Title:  Director 
Institution/Company:  USACEHR 
Address:  568 Doughten Dr, Fort Detrick, MD 21702 
Phone Number:  (301)619-2338 
Email:  rasha.hammamieh1.civ@mail.mil 
 
Name, Rank, and Degree:  Aarti Gautam, PhD  
Title:  Laboratory Chief, 
Institution/Company:  USACEHR 
Address:  568 Doughten Dr, Fort Detrick, MD 21702 
Phone Number:  (301) 619-7683 
Email:  aarti.gautam.civ@mail.mil 
 
1 
A2.  ROLES AND RESPONSIBILITIES 
2 
      
3 
       A2.1  Key Research Personnel   
4 
            Name(s):  Lee M Margolis 
5 
Research Role:  Principal Investigator  
6 
Study Responsibilities:  The Principal Investigator is responsible for the safe and scientifically sound 
7 
conduct of the study.  He will oversee all aspects of study, ensure safety and ethical treatment of 
8 
participants; maintain required documentation for the study and obtain required approvals; and will 
9 
have primary responsibility for data analysis, interpretation, and publication. Dr. Margolis will also be 
10 
actively involved in the volunteer brief, obtaining consent, data collection, performing muscle biopsies, 
11 
catheterization, phlebotomy, and exercise testing and interventions. Drs. Andrew Young, James 
12 
McClung, Scott Montain, or Stefan Pasiakos will be alternate PI in the event of Dr. Margolis’ absence 
13 
and per his written direction.  Dr. Pasiakos will act as the first alternative PI in the event of Dr. Margolis’ 
14 
absence. 
15 
 
16 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
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Name(s):  Andrew J Young, Scott J Montain, James P McClung 
1 
Research Role:  Associate Investigators  
2 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, hypotheses, 
3 
experimental approach and design. Assist PI with volunteer briefs, obtain consent, data collection, 
4 
management, and analysis and manuscript preparation. 
5 
 
6 
Name(s):  Nicholas D. Barringer 
7 
Research Role:  Associate Investigators  
8 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, hypotheses, 
9 
experimental approach and design. Assist PI with volunteer briefs, obtain consent, aerobic exercise 
10 
testing, data collection, management, and analysis and manuscript preparation. 
11 
 
12 
Name(s):  Stefan M Pasiakos 
13 
Research Role:  Associate Investigators  
14 
Study Responsibilities:  Protocol concept development; formulation of protocol questions, hypotheses, 
15 
experimental approach and design. Assist PI with volunteer brief, obtain consent, perform muscle 
16 
biopsies, catheterization, phlebotomy, exercise testing and interventions, data collection, management, 
17 
and analysis and manuscript preparation. 
18 
 
19 
            Name(s):  Marques Wilson 
20 
Research Role:  Project Coordinator  
21 
Study Responsibilities:  Supervise, manage, and coordinate study logistics and biological data 
22 
collection. He will be involved with protocol development and study implementation. He will actively 
23 
participate in data collection to include catheterization, phlebotomy, exercise testing and interventions, 
24 
and DEXA scans.  He will assist in management, analysis and interpretation of data, as well as 
25 
preparation of manuscripts and technical reports for publication. 
26 
 
27 
Name(s):  Claire Whitney and Adrienne Hatch 
28 
Research Role:  Study Dietitian 
29 
Study Responsibilities:  Baseline diet assessments, study diet development, and prepare and 
30 
administer test diets to volunteers.  They will actively participate in data collection to include aerobic 
31 
exercise testing and interventions, and DEXA scans.  She will assist in management, analysis and 
32 
interpretation of data, as well as preparation of manuscripts and technical reports for publication. 
33 
 
34 
       A2.2. Others Involved in the Research, as applicable 
35 
Name(s):  Nancy Murphy  
36 
Research Role:  Biological Sample Coordinator  
37 
Study Responsibilities:  Supervision, management, and coordination of logistics and biological data 
38 
collection. She will be involved with protocol development, study implementation.  Data collection 
39 
responsibilities will involve sample processing. 
40 
 
41 
Name(s):  Christopher Carrigan   
42 
Research Role:  Research Assistant 
43 
Study Responsibilities:   Assist with data collection and biological sample processing. Data collection 
44 
responsibilities will involve with DEXA measurements, phlebotomy, and catheterization. 
45 
 
46 
Name(s):  Nicholas Armstrong  
47 
Research Role:  Research Dietitian 
48 
Study Responsibilities:  Assist with baseline diet assessments, study diet development, and prepare 
49 
and administer test diets to volunteers.  Assist with DEXA measurements and data collection during 
50 
exercise testing. 
51 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
17-17H Glycogen And Microrna Protocol_23MAY2019_V.7_CLEAN 21OCT14 
 
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1 
Name(s):  Laura Lutz 
2 
Research Role:  Research Dietitian 
3 
Study Responsibilities:  Assist with baseline diet assessments, study diet development, and prepare 
4 
and administer test diets to volunteers.  Assist with data collection during exercise testing 
5 
 
6 
Name(s):  Anthony Karis 
7 
Research Role:  Research Assistant 
8 
           Study Responsibilities:  Assist with phlebotomy, catheterization, and sample processing. 
9 
 
10 
Name(s):  Rasha Hammamieh and Aarti Gautam 
11 
Research Role:  Consultants  
12 
           Study Responsibilities:  Drs. Hammamieh and Gautam will receive and analyze coded, de-identified 
13 
muscle RNA for miR Sequencing and bioinformatics analysis.  They will not interact or intervene with 
14 
study volunteers or have access to personal identifiable information.  A coded specimen transfer 
15 
agreement is included with this submission. 
16 
 
17 
A3.  RESEARCH LOCATIONS    
18 
 
19 
USARIEM, Natick MA: The U.S. Army Research Institute of Environmental Medicine (USARIEM) is a DoD 
20 
research facility within the U.S. Army Medical Research and Materiel Command.  It is the Institute 
21 
responsible for conducting basic and applied research to determine the effects of exposure to 
22 
environmental extremes, occupational tasks, physical training, deployment, operational stress and 
23 
nutritional factors on the health and performance of military personnel.  The facility contains environmental 
24 
chambers for controlling temperature and humidity, an environmentally controlled hypobaric chamber, a 
25 
water immersion laboratory, as well as several dry and wet laboratories for animal and human 
26 
experimentation.  The dry laboratories are capable of a broad range of experiments, including 
27 
biomechanical analysis, body composition, energy expenditure, muscle strength and endurance.  The wet 
28 
laboratories include general clinical chemistry analyzers, as well as equipment for ELISA, RIA, histology, 
29 
and molecular biology assays.  Each investigator at the facility has a personal computer with software for 
30 
data management, analysis, presentation and report generation.  Their computers are interfaced with a 
31 
network server for easy, secure data handling and transfer.  Pre-study screening and baseline testing will 
32 
take place at USARIEM for active duty military participants. 
33 
 
34 
 
35 
 
36 
SECTION B:  RESEARCH METHODOLOGY 
37 
 
38 
B1.  ABSTRACT   
39 
 
40 
Understanding how carbohydrate metabolism is altered during exercise and post-exercise with recovery 
41 
nutrition is necessary for the design and implementation of nutrition interventions aimed to maintain 
42 
carbohydrate (e.g., glycogen) stores during military operations.  The primary objective of this investigation 
43 
is to determine the influence of carbohydrate availability (e.g., glycogen depletion and repletion) on skeletal 
44 
muscle microRNA expression, and if changes in circulating microRNA are reflective of changes in skeletal 
45 
muscle microRNA.  microRNA are small non-coding RNA that have been identified as regulators of skeletal 
46 
muscle plasticity in response to exercise, and have recently been found to be measurable stable analytes 
47 
within circulation.  Little is known regarding the influence of substrate availability, particularly carbohydrate 
48 
on microRNA expression in skeletal muscle and circulation.   This randomized cross-over trial will evaluate 
49 
the influence of exercise-induced (cycle ergometry) depletion and diet-induced repletion of skeletal muscle 
50 
carbohydrate stores (e.g., glycogen) on the expression of microRNA in skeletal muscle and circulation in 
51 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
17-17H Glycogen And Microrna Protocol_23MAY2019_V.7_CLEAN 21OCT14 
 
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12 healthy, non-obese, recreational active male and female participants between the ages of 18-39.   After 
1 
a glycogen depletion protocol, volunteers will drink a carbohydrate (CHO: 1.0 g∙kg-1∙hr-1 body mass) or an 
2 
energy free control (CON) beverage during the 3-hr post-exercise recovery phase to assess the initial 
3 
phase of glycogen repletion on microRNA expression.  For the remainder of the day participants will 
4 
consume meals ready-to-eat (MRE) components designed to adequately (6.0 g∙kg-1∙d-1 carbohydrate) or 
5 
inadequately (1.5 g∙kg-1∙d-1 carbohydrate) replenish glycogen stores.  Volunteers will return to the 
6 
laboratory the following day to assess how modified daily carbohydrate intake following glycogen depletion 
7 
influences microRNA expression.  At this time, volunteers will complete 80-min of steady-state (60% 
8 
VO2peak) cycle ergometry, while consuming carbohydrate at a rate of 1.8 g∙min-1 to determine of how 
9 
initiation of exercise with adequate or low glycogen stores effects exogenous carbohydrate efficiency. 
10 
Following a minimum 7-d washout period participants will return to the laboratory to complete the second 
11 
arm of the investigation.  All testing will take place at the USARIEM research labs in Natick, MA. 
12 
 
13 
B2.  BACKGROUND AND SIGNIFICANCE   
14 
 
15 
Carbohydrate Availability and Physical Performance: Carbohydrate is a readily available fuel source in 
16 
skeletal muscle, providing necessary energy to sustain physical performance (1).  The body’s ability to 
17 
store carbohydrate as glycogen is however limited, and can be altered daily by dietary intake, as well as 
18 
exercise type, intensity and duration (2).  Depletion of glycogen stores with prolonged strenuous physical 
19 
activity is associated with fatigue, resulting in reduced work capacity (3).  If glycogen stores are not 
20 
sufficiently replenished during recovery from exercise, physical performance may be diminished during 
21 
subsequent exercise bouts.  As military personnel often engage in sustained physical activity, with mission 
22 
requirements limiting time available to eat, service members can experience degraded ability to sustain 
23 
strenuous physical activity during military operations (4).  Understanding how carbohydrate metabolism is 
24 
altered during exercise and with post-exercise recovery nutrition is crucial for development of nutrition 
25 
interventions aimed to maintain glycogen stores during military operations to mitigate diminished physical 
26 
performance. 
27 
 
28 
Molecular Regulation of Carbohydrate Metabolism: The molecular regulation of carbohydrate 
29 
metabolism in skeletal muscle is a complex network influenced by multiple factors, such as glucose, energy 
30 
availability, muscle contraction, and hormonal environment (e.g., insulin) (5).  Modulation in one or more of 
31 
these factors results in a shift in the cellular environment, which initiates activation (e.g., phosphorylation / 
32 
dephosphorylation) of signaling pathways that control glucose uptake and storage (6).  Specific to exercise-
33 
induced glycogen depletion, diminished glycogen stores stimulate the upregulation of adenosine 
34 
monophosphate-activated protein kinase (AMPK) to enhance glucose uptake and oxidation.  Activation of 
35 
AMPK phosphorylates AKT substrate 160 (AS160) resulting in glucose transporter 4 (GLUT4) translocation 
36 
to the cell membrane to increase glucose uptake (7).  Upregulation of AMPK also stimulate glucose 
37 
breakdown (i.e., glycolysis) through elevated hexokinase activity to increase energy availability in the cell 
38 
(8).  Along with AMPK, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is 
39 
considered a key regulator of energy metabolism (9).  When energy availability is limited within the muscle, 
40 
PGC-1α is upregulated (9).  Activation of PGC-1α modulates the activity of its downstream targets, 
41 
including peroxisome proliferator-activated receptors (PPARs) and estrogen-related receptorα (ERRα), 
42 
which govern fatty acid β-oxidation and carbohydrate oxidation through the tricarboxylic acid cycle (10).  
43 
When there is an increased concentration of circulating glucose and insulin with carbohydrate 
44 
consumption, AMPK and PGC-1α are downregulated to inhibit carbohydrate oxidation and increase 
45 
carbohydrate storage (11).  Elevated insulin concentrations result in insulin binding to its receptor at the 
46 
membrane of cell, resulting in phosphorylation of insulin receptor substrate 1 (IRS1) which stimulates 
47 
GLUT4 translocation to enhance glucose uptake and storage (12).  Together, these molecular processes 
48 
tightly regulate glucose uptake, glycolysis, and fatty acid oxidation to ensure adequate energy availability to 
49 
support cellular function.   
50 
 
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Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
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microRNA and Carbohydrate Metabolism Recently, a new class of small noncoding RNAs, termed 
1 
microRNA, has been identified as critical regulators of energy homeostasis and carbohydrate metabolism 
2 
(13).  Through negative inhibition, microRNA can influence molecular processes by binding to target 
3 
mRNA, resulting in post-transcriptional modifications that repress the translation of protein (14). microRNA-
4 
dependent gene regulation is a complex process, as one microRNA can regulate hundreds to thousands of 
5 
genes (15).  The ability for one microRNA to inhibit the expression of a large number of genes allows a 
6 
single microRNA to repress several genes in a common biological pathway, resulting in robust regulation of 
7 
an entire molecular process (16).  Additionally, one gene can be targeted by multiple microRNA, resulting 
8 
in cooperative / redundant regulation of a signal molecular process (15).  This tight regulation that 
9 
microRNA exert on cellular networks (17) suggests microRNA have may be an important mechanism 
10 
controlling skeletal muscle plasticity (18).   
11 
    
12 
Emerging evidence, suggest that altered expression of microRNA targets specific proteins that regulate 
13 
carbohydrate metabolism.  In cell culture experiments, miR-451 has been shown to target an upstream 
14 
activator of AMPK, resulting in an inhibition of the AMPK signaling cascade (19).  Elevation in miR-451 
15 
expression appears to be sensitive to glucose availability.  When glucose concentrations are low, miR-451 
16 
expression is repressed, allowing for increased activation of AMPK (20).  Conversely, with high glucose 
17 
concentrations miR-451 expression is increased, resulting in diminished AMPK (20).  Similarly microRNA 
18 
have been shown to alter normal insulin signaling processes.  Cell culture experiments have identified miR-
19 
33a/b and miR-126 as inhibitors of IRS (21, 22).  Over expression of these microRNA reduce IRS protein 
20 
content, resulting in insulin resistance and impaired glucose uptake (21, 22).  Furthermore, in disease 
21 
states that altered metabolic demands, such as diet-induced obesity and diabetes, dysregulation of 
22 
microRNA expression has been observed, with specific microRNA (miR-29a, miR-34a, miR-103, miR-107, 
23 
and miR-375) predicted to inhibit signaling proteins associated with insulin signaling upregulated (13).  
24 
Findings from cell culture experiments and disease models indicate that microRNA contribute to regulation 
25 
of carbohydrate metabolism.   
26 
 
27 
Our group recently found that microRNA which are highly enriched in skeletal muscle (i.e., myomiR) are 
28 
sensitive to differing exercise modes with or without essential amino acid plus carbohydrate 
29 
supplementation (unpublished data).  In this investigation we found that weighted endurance-type exercise 
30 
diminished miR-1-3p, miR-206, miR-208a-5p, and miR-499 expression, while conventional cycle ergometry 
31 
endurance exercise increased microRNA expression.  However, when cycle ergometry was combined with 
32 
ingestion of essential amino acids plus carbohydrate, microRNA expression was either downregulated or 
33 
remained the same immediately post and during the recovery phase, compared to baseline values.  
34 
Divergent microRNA were all identified to target insulin signaling, with miR-206, miR-208-5p, and miR-499 
35 
were all predicted to target Akt, an intermediate signaling protein between IRS1 and AS160.  These 
36 
findings suggest that diminished microRNA expression immediately following exercise may have a 
37 
functional role in mediating glucose uptake to replenish glycogen stores.  Corroborating our findings, 
38 
previous investigations have reported the microRNA acutely altered by exercise.  Following an acute bout 
39 
of endurance exercise there is diminished expression of miR-23, while mRNA expression of PGC-1α and 
40 
its downstream targets are upregulated (23, 24).  When miR-23 is overexpressed in skeletal muscle there 
41 
is a reduction in total PGC-1α mRNA and protein content that results in reduced efficiency of mitochondrial 
42 
function, an organelle crucial to cellular energy production (23).  Furthermore, endurance exercise also 
43 
downregulates miR-494, another inhibitor of PGC-1α, allowing for increased gene expression of PGC-1α 
44 
and mitochondrial transcription factor A (25).  While interventions have been conducted examining the 
45 
relationship of microRNA expression to energy sensing genes in response to endurance exercise, no study 
46 
has investigated the influence of altered glycogen availability in response to dietary manipulation on 
47 
microRNA expression.  Understanding how glycogen availability alters expression of microRNA will enable 
48 
determination of their functional role in regulating carbohydrate metabolism to support energy availability to 
49 
fuel physical performance.        
50 
 
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Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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Date:  23 May 2019 
 
 
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Circulating microRNA: Beyond their regulatory function within tissue, microRNA have been reported to be 
1 
stable and reproducible analytes present in circulation (26).  From the cytoplasm, microRNA can be 
2 
released into the circulation in membrane-derived vesicles (exosomes), complexed with proteins, 
3 
lipoproteins (cholesterol), or apoptotic bodies (plasma membrane fragments) (27).  Once released from the 
4 
cell, circulating microRNA can function in cell-to-cell communication, indicating an endocrine-like function 
5 
(28).  Though the exact mechanism by which circulating microRNA influence cellular processes is still 
6 
unknown, it has been observed that alterations in circulating microRNA profiles reflect the underlying 
7 
physiological state of the tissue (29).  In disease states that negative impact skeletal muscle, multiple 
8 
investigations have shown alterations in circulating microRNA profiles are reflective the underlying 
9 
physiological condition of skeletal muscle (30-32).  In healthy individuals, our preliminary findings indicate 
10 
that circulating microRNA profiles can be used as a predictive tool to determine anabolic signaling in 
11 
response to acute resistance exercise in skeletal muscle (33).  Use of advanced statistical and 
12 
bioinformatics analysis identified IGF-1 and mTORC1 signaling as the top canonical pathways upregulated 
13 
post-exercise.  These findings were supported by activation (e.g., phosphorylation) status of mTORC1 
14 
signaling in skeletal muscle following resistance exercise.  Western blotting results determined that an 
15 
upregulation in upstream (p-AktSer473) and downstream (p-70S6K1Thr389) targets of mTORC1, were 
16 
positively associated with increased expression of specific circulating microRNA that have been reported to 
17 
target this pathway.  Though these and other recent (34, 35) findings suggest that circulating microRNA 
18 
profiles are altered by acute exercise, little is known regarding the influence of substrate availability, 
19 
particularly carbohydrate, on circulating microRNA expression.  As microRNA regulate metabolic pathways 
20 
within skeletal muscle, alterations in circulating microRNA expression profiles may be used as a marker for 
21 
underlying molecular adaptions within tissue.  Though assessment of microRNA expression in skeletal 
22 
muscle can yield valuable insight into their functional role in energy metabolism, this type of data collection 
23 
requires invasive muscle biopsies, which is difficult to perform during military field operations.  Identification 
24 
of circulating microRNA as non-invasive markers of carbohydrate metabolism in skeletal muscle has the 
25 
capacity to signal nutritional readiness of service members.   
26 
 
27 
B3.  MILITARY RELEVANCE    
28 
 
29 
Military personnel often engage in sustained physical activity while time to eat and availability of food is 
30 
limited (4, 36, 37). Under such conditions, it is likely that skeletal muscle glycogen stores are depleted, 
31 
reducing the service member’s ability to maintain an optimal level of physical performance. However, 
32 
during ‘real-world’ military training operations there is limited knowledge regarding altered carbohydrate 
33 
metabolism at the skeletal muscle level, as invasive investigations requiring muscle biopsies cannot be 
34 
conducted during training operations.   Identifying the mechanism, as well as determination of non-invasive 
35 
markers of molecular pathways regulating carbohydrate metabolism is critical to move findings from 
36 
controlled laboratory settings to field operations.  Modeling of circulating microRNA may allow for a more 
37 
comprehensive understanding of nutrient adequacy during military operations, allowing for implementation 
38 
of targeted nutrition interventions to optimize military performance.  Support for this protocol comes from 
39 
Defense Health Program (DHP) Research funds, under Joint Program Committee-5 Nutrition and dietary 
40 
supplements working group.     
41 
 
42 
B4.  OBJECTIVES/SPECIFIC AIMS/RESEARCH QUESTIONS   
43 
 
44 
Objectives 
45 
 
46 
1. Determine the influence of carbohydrate availability (e.g., glycogen depletion and repletion) on 
47 
skeletal muscle microRNA expression, and if changes in circulating microRNA are reflective of 
48 
changes in skeletal muscle microRNA. 
49 
 
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Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
Version #:  7 
Date:  23 May 2019 
 
 
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2. Determine how initiation of exercise with adequate or low glycogen stores effects exogenous 
1 
carbohydrate efficiency. 
2 
 
3 
Hypotheses 
4 
 
5 
1. Expression of microRNA in skeletal muscle will be influenced by alterations in glycogen status, and 
6 
that these changes in skeletal muscle microRNA will be associated with modulation in circulating 
7 
microRNA. 
8 
 
9 
2. Exogenous carbohydrate efficiency will be greater when exercise is initiated with lower glycogen 
10 
stores.  Increased efficiency will be associated with higher phosphorylation of IRS-1 and AMPK.    
11 
 
12 
B5.  RESEARCH PLAN   . 
13 
 
14 
       B5.1  Research Design   
15 
 
16 
This study will be a randomized cross-over placebo control trial. 
17 
 
18 
      B5.2  Research Subjects/Population(s) 
19 
 
20 
                B5.2.1  Subject Population(s)   
21 
 
22 
Subject population will be representative of active duty male and female service members, being in good 
23 
health and recreationally active.   
24 
 
25 
                B5.2.2  Number of Subjects, Records, and/or Specimens 
26 
 
27 
To complete testing on the 12 volunteers necessary to reach statistical power, we estimate we will need to 
28 
consent 44 individuals.  All screening will stop once complete data has been collected on 12 volunteers.  
29 
Records and specimen collection are described in the Research Procedures and Data Collection sections.  
30 
During briefings and consenting potential participants will be informed that even though they may be 
31 
eligible and want to participate, if we are able to obtain enough data from preceding subjects, they may not 
32 
ultimately be tested.      
33 
     
34 
     B5.2.3  Inclusion Criteria 
35 
 
36 
• 
Men and women aged 18 – 39 years 
37 
• 
Weight stable (±5 lbs) for at least 2 months prior to the start of the study 
38 
• 
Body mass index (BMI) between 18.5-30 kg/m2 
39 
• 
Recreationally active based on assessment of physical activity history 2-4 days per week aerobic 
40 
and/or resistance exercise 
41 
• 
Refrain from taking any NSAIDS (i.e., aspirin, Advil®, Aleve®, Naprosyn®, or any aspirin-containing 
42 
product for 10 days before and at least 5 days AFTER each muscle biopsy. (*Tylenol® or 
43 
acetaminophen is ok to use if needed for discomfort) 
44 
• 
Refrain from the use of alcohol and nicotine for the duration of the study  
45 
• 
Females must be on contraception (e.g., oral birth control, NuvaRing®, Depo Provera®, etc.) 
46 
• 
Supervisor approved leave status for federal civilian employees working within the US Army Natick 
47 
Soldier Systems Center 
48 
 
49 
    B5.2.4  Exclusion Criteria 
50 
 
51 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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 
Metabolic or cardiovascular abnormalities, gastrointestinal disorders (i.e., kidney disease, diabetes, 
1 
cardiovascular disease, etc.)  
2 
 
Disease or medication (i.e., diabetes medications, statins, corticosteroids, etc) that affects 
3 
macronutrient utilization and/or the ability to participate in strenuous exercise  
4 
 
Allergies or intolerance to foods (including but not limited to lactose intolerance/milk allergy), vegetarian 
5 
practices, or medications (including, but not limited to, lidocaine or phenylalanine) to be utilized in the 
6 
study 
7 
 
Anemia (HCT < 38) and Sickle Cell Anemia/Trait 
8 
 
Abnormal PT/PTT test or problems with blood clotting  
9 
 
Present condition of alcoholism, use of nutritional/sports supplements, anabolic steroids, or other 
10 
substance abuse issues  
11 
 
Musculoskeletal injuries that compromise the ability to exercise 
12 
 
Blood donation within 8 weeks of beginning the study 
13 
 
Pregnancy 
14 
 
15 
      B5.3  Research Procedures   
16 
 
17 
Study Design 
18 
Following a 10-hr overnight fast, a baseline blood draw will be conducted.  A baseline muscle biopsy, 
19 
obtained from the lateral portion of the vastus lateralis, will be collected under local anesthetic (lidocaine).  
20 
Participants will rest for 15-min before beginning a 5-min warm-up on a cycle ergometer at 50% peak 
21 
power output (determined from screening VO2peak test).  A glycogen depletion protocol (38) will then be 
22 
initiated with participants cycling at various intensities until failure.  A second muscle biopsy will be 
23 
performed immediately after completion of glycogen depletion.  This biopsy will allow for confirmation of 
24 
glycogen depletion and allow for the assessment of the initial impact of exercise-induced glycogen 
25 
depletion on microRNA expression.  Participants will then consume a carbohydrate (CHO: 1 g∙kg-1∙hr-1) or 
26 
an energy free control (CON) beverage matched for taste and color during the first 3-hr post glycogen 
27 
depletion.  Blood will be drawn from a catheter every 30-min post-exercise to assess changes in serum 
28 
glucose and insulin.  At 3-hr post-exercise a third muscle biopsy will be conducted to assess acute effects 
29 
of carbohydrate consumption on microRNA during the initial phase of glycogen resynthesis during the early 
30 
phase recovery from exercise.    For the remainder of the day participants will consume meals ready-to-eat 
31 
(MRE) components designed to adequately or inadequately replenish glycogen stores.  On the study day in 
32 
which participants receive the carbohydrate beverage, daily intake will be provided as 6.0 g∙kg-1 
33 
carbohydrate, 1.2 g∙kg-1 protein, 1 g∙kg-1 fat.  On the study day in which participants receive the placebo 
34 
beverage, daily intake will be provided as 1.5 g∙kg-1 carbohydrate, 1.2 g∙kg-1 protein, 3.0 g∙kg-1 fat.  
35 
Following a 10-hr overnight fast, participants will return to the laboratory for a fourth muscle biopsy.  As 24-
36 
hrs is sufficient time to replenish glycogen stores (1), the fourth muscle biopsy with modified daily 
37 
carbohydrate intake will allow for isolation of the effect of carbohydrate availability on microRNA 
38 
expression.  Volunteers will then complete 80-min of steady-state (~60% VO2peak) cycle ergometry 
39 
consuming carbohydrate at a rate of 1.8 g∙min-1 enriched with U-13C-glucose and U-13C6-D-fuctose  to 
40 
determine exogenous carbohydrate efficiency.  A final biopsy will be taken at the conclusion of the exercise 
41 
bout. This fifth biopsy will be conducted to assess the impact of initiating steady-state exercise with 
42 
adequate or low glycogen stores on molecular markers of glucose uptake and substrate utilization.  
43 
Following a minimum 7-d washout period volunteers will return to the laboratory to complete the second 
44 
arm of the investigation.  To ensure glycogen stores are similar between volunteers on testing day, 48-hrs 
45 
prior to testing all volunteers will complete a glycogen depletion protocol and then consume a diet providing 
46 
6.0 g∙kg-1 carbohydrate to replenish glycogen stores.     
47 
 
48 
 
49 
 
50 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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1 
Figure 1. Study Design 
2 
 
3 
 
4 
 
5 
Research Procedures 
6 
Diet Intervention 
7 
Volunteers will complete a baseline 3-d diet record (Appendix A) and a 3-d activity log (Appendix B) 
8 
according to instructions provided by study team dietitians (39).    The data will be analyzed using Food 
9 
Processor SQLTM (Salem, OR Version 10.0) and the American College of Sports Medicine (ACSM) 
10 
Compendium of Physical Activities, respectively.  Information for these forms will be collected to estimate 
11 
volunteers total daily energy requirements.  To ensure all volunteers begin the protocol day with similar 
12 
glycogen stores, following glycogen depletion 48-hrs before testing, volunteers will be provided with food 
13 
and beverages (except water) containing 6.0 g carbohydrate ∙kg-1∙d-1 to replete glycogen stores.  Meals will 
14 
be derived from military combat rations (MRE) and supplemental food items.  Example of energy and 
15 
macronutrient content will be consistent with ACSM dietary guidelines (Table 1) (40).         
16 
 
17 
On glycogen depletion protocol days, volunteers will receive standardized meals derived from MRE 
18 
components and supplemental carbohydrate-based food items.  Meals will either provide adequate 
19 
carbohydrate (6.0 g∙kg-1∙d-1) to replenish glycogen stores or provide low carbohydrate (1.5 g∙kg-1∙d-1) so 
20 
glycogen stores remain below baseline concentrations (Table 1).  Study dietitians will prepare and 
21 
administer meals to volunteers.  In this cross-over study the order of carbohydrate intake amount will be 
22 
randomized to avoid order bias.    
23 
 
24 
 
25 
 
26 
 
27 
 
28 
 
29 
 
30 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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Table 1. Energy and Macronutrient Intake for Lead-in Diet and Protocol Days 
1 
 
Lead in Diet 
Protocol Days 
 
 
Adequate 
CHO 
Low CHO 
Energy (kcal∙d-1) 
3024 
3024 
3024 
Protein (g∙kg-1∙d-1, kcal∙d-1) 
1.2 (384) 
1.2 (384) 
1.2 (384) 
Carbohydrate (g∙kg-1∙d-1, kcal∙d-1) 
6.0 (1,920) 
6.0 (1,920) 
1.5 (480) 
Fat (g∙kg-1∙d-1, kcal∙d-1) 
1.0 (720) 
1.0 (720) 
3.0 (2160) 
Values based on an 80 kg individual 
2 
 
3 
Volunteers will be instructed to maintain their normal physical activity during the study.  However, 
4 
volunteers will be required to refrain from any additional physical activity outside of the study (resistance 
5 
and endurance exercise) 48-hrs before the glycogen depletion protocol days.  Restriction of physical 
6 
activity will minimize complications and the potential for carryover effects that may influence study 
7 
outcomes.   
8 
 
9 
Anthropometric Data 
10 
Anthropometrics, performed using standardized techniques and equipment, will be used to determine 
11 
volunteer eligibility and characterize study participants.  Height will be measured to the nearest 0.1 cm 
12 
using a stadiometer at screening. Body mass will be measured, semi-nude and after an overnight fast (≥ 8 
13 
hr), using a calibrated digital scale to the nearest 0.1 kg at screening.  Body mass will be measured at 
14 
baseline, the morning of and the morning after glycogen depletion protocol days. 
15 
 
16 
Body composition will be determined using dual energy x-ray absorptiometry (DEXA, DPX-IQ, GE Lunar 
17 
Corporation, Madison, WI).  The DEXA technique allows for the non-invasive assessment of soft tissue 
18 
composition by region with a precision of 1-3% (41). The volunteer will lay face-up on the DEXA 
19 
densitometer table in shorts, t-shirts, and stocking feet.  Volunteers will be asked to remain motionless for 
20 
the 8-10 min scan. These data will be used to calculate total body mass, fat-free mass, and fat mass. 
21 
Calibration to external standards will be performed before actual data collection. The operator remains in 
22 
the room with the volunteer during the scan. 
23 
 
24 
Determination of Peak Oxygen Uptake 
25 
Peak oxygen uptake (VO2peak) will be determined by a cycle ergometer test using a computer-based 
26 
metabolic system (True Max 2400, Parvomedics, Sandy, Utah, USA).  Volunteers will be instructed to fast 
27 
overnight (≥ 8 hr) before testing.  Volunteers will be clothed in appropriate athletic attire and perform this 
28 
assessment at standard ambient indoor temperature (20-22C) and humidity conditions (30-80%).  
29 
Following instructions on testing procedures, the volunteer will be allowed a 5-min warm-up pedaling at 
30 
70W.   At the start of testing, the volunteer will put on a nose clip and a mouthpiece connected to a 2-way 
31 
respiratory valve, which is attached to a head piece to hold it in place.  Every minute, workload intensity will 
32 
be progressively increase by 30W until the volunteer is fatigued or unable to maintain a pedaling rate that 
33 
either maintains or increases O2 consumption.   Heart rate will be monitored using a heart-rate monitor 
34 
(Polar Electro Inc, Oulu, Finland) the last 30 seconds of each workload.  The test will be stopped 
35 
immediately if the subject reports angina-like symptoms, exertional syncope, shows signs of poor perfusion 
36 
(i.e., light-headedness, confusion, ataxia, pallor, cyanosis, nausea, or cold and clammy skin), or if testing 
37 
equipment fails.   
38 
 
39 
Glycogen Depletion Protocol 
40 
The glycogen depletion protocol will be completed on a cycle ergometer, with intensity based on results 
41 
from VO2peak assessment.  Volunteers will begin with a 5-min warm-up at 70 watts before beginning the 
42 
glycogen depletion protocol.  After the warm-up period volunteers will complete 2-min of high-intensity 
43 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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cycling (work period) at 90% VO2peak, followed by 2-min recovery period where volunteers will cycle at 50% 
1 
VO2peak.  This work-to-recovery ratio will be maintained until volunteers are no longer able to complete 2-
2 
min of cycling at 90% VO2peak, determined as the inability to maintain a cycling cadence of 60 rpm for 15-
3 
sec.  Cycling intensity will then be lowered to 80% VO2peak.  When the volunteer is unable to complete 2-
4 
min of cycling at 80% VO2peak, cycling intensity will be lowered to 70% VO2peak.   Once the volunteer can no 
5 
longer complete 2-min of cycling at 70% VO2peak, cycling intensity will be lowered to 60% VO2peak.   The 
6 
glycogen depletion protocol will be terminated once the volunteer is unable to complete 2-min of cycling at 
7 
60% VO2peak.  For each drop in the intensity of the work period, the cycling intensity during the recovery 
8 
period will be maintained at 50% VO2peak for 2-min.  This exercise protocol will maximally deplete muscle 
9 
glycogen stores (38).  Prior to the protocol day, volunteers will perform two practice sessions to ensure 
10 
they are familiar with the glycogen depletion protocol procedures.  Following familiarization volunteers will 
11 
complete four glycogen depletion protocols, two 48-hrs before testing and two testing days. 
12 
 
13 
Volunteers will be allowed to consume water ad libitum during the glycogen depletion protocol.  Exercise 
14 
will be conducted in a temperature controlled room.  Heart rate will be monitored using a heart-rate monitor 
15 
and the session will be terminated if the subject reports any discomfort (i.e. angina-like symptoms or 
16 
exertional syncope) or shows signs of poor perfusion, or if there is an equipment failure.   
17 
 
18 
Muscle Biopsy  
19 
Percutaneous muscle biopsy will be performed to obtained skeletal muscle samples for assessment of 
20 
muscle glycogen, microRNA expression and molecular pathways regulating carbohydrate metabolism.  
21 
Muscle samples will be taken from the vastus lateralis using a 5-mm Bergstrom needle with manual suction 
22 
while the participant is under local anesthesia (1% lidocaine) according to the approved USARIEM SOP 
23 
(42, 43). Multiple passes with the biopsy needle may be necessary to obtain adequate sample with each 
24 
biopsy.  Muscle biopsies will be conducted immediately before and after the muscle glycogen depletion 
25 
protocol.  After the second muscle biopsy volunteers will rest and consume either a carbohydrate beverage 
26 
or a matched placebo.  Three hours following the conclusion of the glycogen depletion protocol a third 
27 
biopsy will be obtained to assess the acute influence of carbohydrate intake.  Volunteers will be allowed to 
28 
leave at this point.  Volunteers will return the following morning for a fourth muscle biopsy taken at a new 
29 
incision site.  In this same incision a fifth biopsy will be taken after 80-min of steady state exercise to 
30 
determine the influence of adequate or low glycogen storage on exogenous carbohydrate uptake and 
31 
oxidation during exercise.  Following a minimum 7-d washout period, volunteers will return to the laboratory 
32 
to complete the second arm of the investigation.  A total of ten muscle biopsies will be conducted per 
33 
volunteer.          
34 
 
35 
Assessment of Resting Respiratory Quotient  
36 
Respiratory Quotient (RQ) will be measured under resting conditions before the 24-hr post glycogen 
37 
depletion muscle biopsy, using open circuit indirect calorimetry (Parvo Medics).  Assessment of RQ will 
38 
allow us to determine the influence of glycogen storage on carbohydrate and fat oxidation.  Measurements 
39 
will occur between 0500-0800-hrs following a minimum 10-hr overnight fast. Volunteers will rest in the 
40 
supine position for approximately 30-min prior to each measurement in a quiet and dim, temperature 
41 
regulated room. To minimize error, volunteers will be instructed to minimize movement once the 
42 
mouthpiece is inserted, or the mask/hood is placed over their heads to collect expired air. The test will be 
43 
discontinued when 10-min of steady state oxygen consumption (VO2) and carbon dioxide production 
44 
(VCO2) are recorded.    
45 
 
46 
Steady-State Exercise 
47 
After the fourth muscle biopsy volunteers will complete 80-min of steady-state exercise on the cycle 
48 
ergometer at 60 ± 5% of their VO2peak.  During exercise VO2 and VCO2 will be measured for 4-min using a 
49 
metabolic cart (Parvo Medics) at approximately 0, 15, 30, 45, 60, and 75-min to determine substrate 
50 
oxidation. HR (measured continuously; Polar Electro) will be recorded at the same times as the metabolic 
51 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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cart measurements. Breath samples will be collected at approximately 0, 20, 40, 50, 60, 65, 70, 75, and 80-
1 
min to determine exogenous carbohydrate oxidation using single-patient breath collection bags (Quin-Tron 
2 
Instrument Company, Milwaukee, WI, USA). Breath will be transferred to 20 mL evacuated tubes.    
3 
 
4 
Carbohydrate Supplementation 
5 
After the post glycogen depletion muscle biopsy, volunteers will drink a CHO (glucose and fructose in a 2:1 
6 
ratio) or CON beverage (Combat Feeding, Natick, MA.  Carbohydrate will be provided at 1 g∙kg-1∙hr-1 in 
7 
accordance with current American College of Sports Medicine Guidelines (40). The CON beverage will be 
8 
sugar free and void of other nutrients to allow for assessment of the independent effect of carbohydrate 
9 
availability on skeletal muscle and circulating microRNA expression.  Beverages will be consumed 
10 
immediately following the post glycogen depletion muscle biopsy and then every 30 min thereafter (5 
11 
times) until the 3-hr post glycogen depletion muscle biopsy.  
12 
 
13 
Additionally, all participants will consume the CHO beverage at 1.8 g∙min-1 during steady-state exercise to 
14 
examine the impact of glycogen availability on exogenous carbohydrate oxidation efficiency.  Volunteers 
15 
will consume the beverage immediately before the start of the exercise and then again at 20, 40, and 60-
16 
min during the exercise bout.  The CHO beverage consumed during exercise will be enriched with 200 mg 
17 
of U-13C-glucose and U-13C6-D-fructose (Cambridge Isotope Laboratory, Andover, MA, USA) to significantly 
18 
increase the isotopic enrichment above natural levels and optimize the measurement of exogenous 
19 
carbohydrate oxidation.     
20 
       
21 
B5.4  Data Collection   
22 
 
23 
Blood Sampling 
24 
Blood samples will be collected using intravenous access by a USARIEM credentialed phlebotomist on 
25 
glycogen protocol days at baseline (before glycogen depletion), immediately following and every 30 min 
26 
after the glycogen depletion protocol to assess circulating glucose and insulin (Figure 1).  Baseline and 24-
27 
hr post glycogen depletion blood sampling will occur after a 10-hr overnight fast.  A total of 120 mL of blood 
28 
will be sampled from each volunteer during each glycogen depletion protocol day study (Table 2).  Blood 
29 
samples will be used to assess nutrient status (glucose and insulin) and circulating microRNA.     
30 
   
31 
Biochemical indices of metabolic homeostasis will be measured from stored samples at USARIEM.  
32 
Unused serum will be retained for future use if subject provide permission in the informed consent 
33 
document.  Samples will be stored at USARIEM using subject identification numbers. 
34 
 
35 
Table 2. Volume of Blood Sampled for Glycogen Protocol Day 
36 
Tube 
Number of Tubes 
Total Volume (mL) 
10 mL Red Top Tube 
5 
50 
7 mL Red Top Tube 
10 
70 
Total 
15 
120 
 
37 
Breath Sample Processing for Isotopic Enrichment 
38 
Measurement of 13C/12C in expired CO2 will be measured using isotope-ratio mass and cavity ring-down 
39 
spectroscopy (Metabolic Solutions, Inc., Nashua, NH). 
40 
 
41 
Calculations of Carbohydrate and Fat Oxidation 
42 
Fat and CHO oxidation will be calculated from VO2 and VCO2 measured using open circuit indirect 
43 
calorimetry (4 min collection), neglecting the contribution of protein oxidation to the energy yield (44): 
44 
 
45 
Fat oxidation (g/min) = 1.695 x VO2 (L/min) – 1.701 x VCO2 (L/min)  
46 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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Glucose oxidation (g/min) = 4.585 x VCO2 (L/min) – 3.226 x VO2 (L/min) 
1 
 
2 
Calculations of Exogenous and Endogenous Glucose Oxidation 
3 
Exogenous glucose oxidation can be calculated: 
4 
 
5 
Exogenous glucose (g/min) = VCO2 [(Rexp - Rref)/(Rexo - Rref)]/k 
6 
 
7 
where VCO2 is in L/min, Rexp is the observed isotopic composition of expired CO2, Rref is the isotopic 
8 
composition of expired CO2 at rest before ingestion of the first dose of 13C-glucose and U-13C6-D-fructose, 
9 
Rexo is the isotopic composition of the exogenous glucose ingested, and k (0.747 L/g) is the volume of 
10 
CO2 provided by the complete oxidation of glucose. Endogenous glucose oxidation can be calculated by 
11 
subtracting exogenous glucose oxidation from total CHO oxidation. The first 40 min of steady-state 
12 
exercise will allow for equilibration between the 13C/12C in expired CO2 and the 13C/12C in CO2 produced in 
13 
tissues (45). Thus, endogenous glucose oxidation will only be calculated from samples obtained in the last 
14 
40 min of steady-state exercise (40 to 80 min). 
15 
 
16 
Muscle Glycogen 
17 
Approximately 20 mg of muscle will be dehydrated in a freeze dryer.  Samples will then be ground to 
18 
powder and visible connective tissue will be removed.  Powdered muscle will be placed in 500 µl 2 N HCl.  
19 
Samples will then be placed in an incubator for 120 min at 100˚C.  Following incubation samples will be 
20 
neutralized with 1500 µl 0.67 N NaOH.  Muscle glycogen will be in solution at this point.  Glycogen will be 
21 
quantified by a fluorometric assay (Sigma-Aldrich, St. Louis, MO, USA).      
22 
 
23 
Intramuscular Triglyceride Concentrations 
24 
Intramuscular triglyceride concentrations will be determined using the Folch method (46). Dehydrate 
25 
muscle will be homogenized in a 2:1 chloroform-to-methanol solvent. Following extraction, samples will be 
26 
saponified in ethanolic KOH at 60 °C, and glycerol content will determined using a commercially available 
27 
colorimetric assay (Sigma) 
28 
 
29 
mRNA and microRNA Expression 
30 
Total RNA will be isolated from approximately 25 mg of muscle using a mirVanaTM miRNA isolation kit 
31 
(Invitrogen, Carlsbad, CA, USA).  Quantity and quality of RNA will be assessed using a Nanodrop ND-
32 
1000spectrophotometer (Nanodrop, Wilmington, DE, USA).  Equal amounts of total RNA will be 
33 
synthesized into cDNA for analysis of mRNA (iScriptTM Advanced cDNA Synthesis Kit; Bio-Rad) and a 
34 
TaqMan® microRNA RT kit (Applied Biosystems, Foster City, CA, USA).  Individual primers will be used to 
35 
determine the mRNA expression of known intracellular targets regulating muscle metabolism, to include 
36 
but not limited to PGC-1α, SIRT1, ACC, AMPK, PDK4, IRS1, and GLUT4.    miRNA (20-40 bp) will be size-
37 
selected from total RNA using Illumina’s protocol (US Army Center for Environmental Health Research, 
38 
Frederick, MD). The sorted miRNA will be tagged and assayed using the Illumina NextSeq instrument. 
39 
Briefly, 500 ng total RNA will be used to construct sequencing libraries. Subsequently, the samples will be 
40 
amplified using index-tagged primers to facilitate multiplexing. The amplified cDNA constructs will be size-
41 
selected, quality controlled before loading to the NextSeq instrument to generate 50 base reads. Image 
42 
analysis and base calling will be performed using the Illumina pipeline.  
43 
 
44 
Preprocessing of raw base calls, sample de-multiplexing, trimming and filtering will be similar to that 
45 
described in ‘Analysis of sequencing output’. For the miRNA quantification/enrichment, reads are first 
46 
aligned to the Rfam RNA database to filter and profile other small RNA species in the samples. Then reads 
47 
are mapped for small RNA annotation against reference zebrafish mature mature RNA sequences from the 
48 
miRBASE database. The miRNA read count matrix for each sample will be generated using open source 
49 
tools such as PICARD.   
50 
 
51 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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Following identification of skeletal muscle microRNA that had a significant change, Taqman® probes 
1 
(Applied Biosystems) will be used to assess the expression of these microRNA in serum to determine their 
2 
potential use as noninvasive markers of altered carbohydrate metabolism following glycogen depletion and 
3 
repletion.  Total circulating miRNA will be extracted from 200 µL serum using miRNeasy Serum/Plasma kit, 
4 
which allows for extraction and purification of small (< 200 nt) cell-free RNA (Qiagen, Valencia, CA, USA). 
5 
Exosomal circulating miRNA will also be extract from 1000 µL serum using miRCURYTM RNA Isolation Kit 
6 
to measure miRNA. To avoid introduction of potentially contaminating material, prior to RNA extraction 
7 
serum samples will be centrifuged for 10 min at 4°C to remove cellular debris.  Supernatant will be 
8 
removed and transferred to a new tube without disturbing the pellet.  Due to the small amount of RNA in 
9 
the serum, 3.5 µL of a Spike-In Control (C. elegans miR-39; Qiagen) will be added to all samples prior to 
10 
extraction of RNA to determine the yield of template recovered.  After extraction 3 μl of serum RNA will 
11 
reverse transcribed using the TaqMan® microRNA RT kit (Applied Biosystems) with miRNA-specific stem-
12 
loop RT primers pooled in 1X-Tris-EDTA (TE) buffer for a final dilution of 0.05X.  A pre-amplification step 
13 
will be performed after reverse transcription to increase cDNA template using a primer pool of 20 X 
14 
Taqman® Small RNA Assays (Applied Biosystems) for miRNA of interest at 0.05X concentration in 1X TE 
15 
buffer.  All serum miRNA will be normalized to the geometric of external (Spike-In Control C. elegans miR-
16 
39) and internal controls to allow for both technical and inter-individual normalization (47). Geometric mean 
17 
of controls will be used to correct for possible outlying values and abundance differences between the 
18 
different controls (48).   
19 
 
20 
All reverse transcription for mRNA and miRNA, and pre-amplification of serum miRNA will be conducted in 
21 
a T100TM Thermal Cycler (Bio-Rad, Hercules, CA). A StepOnePlusTM real-time PCR system (Applied 
22 
Biosystems) will be used to perform all mRNA and miRNA analysis.  Fold changes will be calculated using 
23 
the ∆∆ cycle threshold (∆∆CT) method as described below in statistical analysis section. 
24 
 
25 
Bioinformatics Analysis 
26 
microRNA with significant changes in their expression will be uploaded to DNA Intelligent Analysis 
27 
(DIANA)-miRPath 3.0 (Alexander Fleming Biological Sciences Research Center [BSRC], Athens, Greece; 
28 
http://diana.cslab.ece.ntua.gr) to determine potential molecular pathways that these microRNA have 
29 
previously been reported to regulate. Relevant Kyoto Encyclopedia of Genes and Genomes (KEGG; 
30 
http://www.genome.jp/kegg/) pathways will be identified using experimentally verified targets from TarBase 
31 
7.0 (Alexander Fleming BSRC).  Based on findings from this analysis, relevant gene and protein 
32 
expression of relevant targets will be assessed.    
33 
 
34 
Western Blotting 
35 
Approximately 30 mg of muscle will be homogenized in ice-cold buffer (1:10 wt/vol) containing 50 mM Tris–
36 
HCl (pH 7.5), 5 mM Na-pyrophosphate, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 10% glycerol (v/v), 1% 
37 
Triton-X, 1 mM DTT, 1 mM benz-amidine, 1 mM PMSF, 10 μg mL−1 trypsin inhibitor and 2 μg mL−1 
38 
aprotinin.  Homogenate will be centrifuged for 15 min at 10,000 × g at 4°C.  Protein concentration of 
39 
supernatant (lysate) will be determined using 660 nm Protein Assay (ThermoFisher Scientific, Waltham, 
40 
MA, USA).  Phosphorylation status and total protein expression of molecular markers associated with 
41 
carbohydrate metabolism will be determined using Western blot.  Muscle lysates will be solubilized in 
42 
Laemmli buffer, with equal amounts of total protein (15 μg) separated by SDS-PAGE using precast 
43 
Tris·HCl gels (Bio-Rad). Proteins will be transferred to polyvinylidene fluoride (PVDF) membranes and 
44 
exposed to commercially available primary antibodies of intracellular markers involved with muscle 
45 
metabolism to include but not limited to Akt, p-AktSer473, AMPKα, p- AMPKαThr172, PGC-1α , SIRT1, ACC, p-
46 
ACCSer79, PDK4, IRS1, p-IRS1 Ser302, GSK-3β, p-GSK-3βSer9, GSK-3α , p-GSK-3α Ser21, GLUT4 and PEPCK 
47 
(Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight.  Labeling will be performed using 
48 
secondary antibody (anti-rabbit IgG conjugate with horseradish peroxidase; Cell Signaling Technology), 
49 
and chemiluminescent reagent will be applied (Super Signal, West Pico Kit; Pierce Biotechnology, 
50 
Rockford, IL, USA). Blots will be quantified using a phosphoimager (ChemiDoc XRS; Bio-Rad) and Image 
51 

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Lab software (Bio-Rad). To confirm equal protein loading per well a normalizing protein such as GAPDH or 
1 
β-actin will be assessed.  
2 
 
3 
Citrate Synthase Activity 
4 
Homogenate from muscle samples prepared for Western blotting will be used to assess citrate synthase 
5 
activity. Enzyme activity will be determined using a colorimetric assay analyzed on an SpectraMax® M 
6 
Series Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA), by combining 10 μl of 
7 
diluted (1:10; 0.1 M Tris HCl pH 8.1) sample to 150 μl of reaction master mix (1 mL DNTB, 3 mg Acetyl 
8 
CoA, and 8 mL 0.1 M Tris HCl pH 8.1). The reaction was initiated when 10 μl of 10 mM oxaloacetate was 
9 
added to each well (49). Samples were read at 412 nm. Data were normalized to protein content. 
10 
 
11 
Any use of the data or samples outside of this defined research plan will be submitted as a protocol 
12 
amendment or a new protocol. 
13 
 
14 
      B5.5  Managing Data and/or Human Biological Specimens for this Research   
15 
 
16 
All data and medical information obtained will be considered privileged and held in confidence.  Study 
17 
volunteers will be assigned unique subject identification (ID) numbers that will not contain any personal 
18 
identifiers such as name, social security number, address, date of birth, zip code, etc.  This study subject 
19 
ID number will be used on all data collection instruments, to include questionnaires, data collection 
20 
forms(Appendix D-F), computer records, etc.  A number will be assigned as each volunteer is medically 
21 
cleared for participation.  A master list linking the volunteers’ names and ID numbers will be kept in a 
22 
separate locked file in the principal investigator's or project manager’s office, or kept in a computer file with 
23 
password-protected access restricted to the principal investigator and project manager.  When the results 
24 
of the research are published or discussed in conferences, no information will be included that would reveal 
25 
identity.  De-identified muscle RNA samples for miR sequencing will be shipped USACEHR and breath 
26 
samples for isotopic analysis will be shipped to Metabolic Solutions via FedEx and stores in these 
27 
laboratories until analyzed.  Once analyzed, there will be no remaining sample for storage. All other study 
28 
samples will be analyzed at USARIEM and stored in n -80ºC freezer at USARIEM in room 322 or 304.  All 
29 
samples will be stored using the subject identification number. 
30 
 
31 
Only personnel assigned to the research study by the principal investigator will have access to the data.  
32 
Hard copy data records will be stored for a minimum of three years from the time the study is completed.  
33 
Electronic data records will be maintained for a period of at least five years after the study has been 
34 
completed.   
35 
 
36 
      B5.6  Managing Data and/or Human Biological Specimens for Future Research 
37 
 
38 
Any use of the samples outside of this defined protocol will be submitted as a protocol amendment or a 
39 
new protocol.  Samples will be retained for future analyses once the protocol is closed if the subject 
40 
provided permission in the informed consent document.   
41 
 
42 
     B5.8  Statistical Analysis 
43 
 
44 
               B5.8.1  Sample Size Estimation 
45 
 
46 
Statistical power and sample size were determined using the minimum fold change required for a 
47 
physiologically relevant difference in microRNA expression.  Variance (standard deviation) was based 
48 
on recent publications assessing change in microRNA expression in skeletal muscle (18, 50, 51) and 
49 
circulating (34, 35, 52) in response to an acute bout of exercise.  Using this information the sample 
50 
size necessary to determine statistical significance between intervention arms, with a mean fold 
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change of 1.5 ± 1 is 9 per group, with an alpha of 0.05 and 80% power.  In the present study, 12 
1 
volunteers will be studied in a repeated measures crossover design to account for potential greater 
2 
inter-individual variability.     
3 
 
4 
               B5.8.2  Data analysis   
5 
 
6 
Statistical analyses will be conducted using either SPSS (IBM Corp. Armonk, NY), SAS 9.3 (SAS 
7 
Institute Inc., Carey, NC), or equivalent.  Common descriptive statistics will be used to describe 
8 
volunteer characteristics.  Shapiro-Wilk tests will be used to determine normality of data.  Mixed 
9 
model repeated measures ANOVA will be performed to determine main effects of time (baseline, 
10 
immediately, 3-hr, and 24-hr post glycogen depletion), carbohydrate intake (6.0 g·kg-1·d-1 versus 1.5 
11 
g·kg-1·d-1) and time-by-carbohydrate interactions for skeletal muscle and circulating microRNA, mRNA 
12 
expression, and markers of nutritional status.  If interactions are significant, Bonferroni correction will 
13 
be used to examine these relationships.  Correlation coefficients and multiple regression analysis will 
14 
be used to evaluate relationships between study outcome measures.  The alpha level will be adjusted 
15 
for multiple comparisons, with the level for statistical significance set at P < 0.05.   
16 
 
17 
 
18 
 
19 
SECTION C:  HUMAN RESEARCH PROTECTIONS 
20 
 
21 
C1.  RECRUITMENT AND CONSENT   
22 
 
23 
       C1.1  Identification and Selection of Subjects   
24 
 
25 
Potential volunteers will be recruited from the U.S. Army Natick Soldier Center pool of military volunteers 
26 
during a face-to-face briefing as described in the below Recruitment Process section.  Additional volunteers 
27 
will be recruited from civilian population and active duty military stationed at the U.S. Army Natick Soldier 
28 
Center, as well as civilians from the local surrounding area using flyers and electronic posting (Appendix 
29 
G) 
30 
 
31 
       C1.2  Recruitment Process   
32 
 
 
33 
Superiors of Service members (e.g., unit officers, senior NCOs, and equivalent civilians) / supervisors of 
34 
DoD civilians (e.g., military and civilian supervisors or anyone in the supervisory structure) shall not be 
35 
present at any recruitment sessions or during the consent process in which members of units under their 
36 
command / personnel under their supervision are afforded the opportunity to participate as human subjects 
37 
of research.   
38 
 
39 
For Soldiers in the U.S. Army Natick Soldier Center pool of military volunteers, the Principal Investigator 
40 
will furnish a copy of the consent form to the Human Research Subjects Program Coordinator or designee. 
41 
The Coordinator will schedule the consent briefing for the military human research volunteer platoon and 
42 
will serve as ombudsman during the briefing.  Civilians and permanent party military will be recruited for the 
43 
study using posted or electronically distributed or though informational briefs.  Principal Investigator or 
44 
Project Coordinator will receive and respond to inquiries submitted from these recruitment materials, and 
45 
will schedule informed consent briefings for these potential volunteers. Potential volunteers not in the U.S. 
46 
Army Natick Soldier Center pool of military volunteers will be briefed one-on-one by the Principal 
47 
Investigator or Project Coordinator. 
48 
 
49 
       C1.3  Eligibility   
50 
 
51 

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All potential volunteers will complete the background questionnaire (Appendix H). Volunteers must be 
1 
medically cleared by the Office of Medical Support and Oversight (OMSO) for participation in accordance 
2 
with USARIEM procedures outlined for screening volunteers for research involving exercise. Potential 
3 
military and civilian participants will undergo the same clearances used at USARIEM for research involving 
4 
maximal aerobic exercise testing. In addition, volunteers will be screened for problems with blood clotting, 
5 
including prothrombin time (PT)/ partial thromboplastin time (PTT), which is a specific criterion for research 
6 
involving muscle biopsies. Health problems identified during the screening process will be documented and 
7 
a copy provided to the volunteer. The volunteer will be encouraged to make an appointment with their 
8 
primary care provider for a full evaluation of the problem. Volunteers with evidence of any physical, mental, 
9 
and/or medical conditions that would make the proposed studies relatively more hazardous will be 
10 
excluded. Any personal health information collected during this screening process will be destroyed at the 
11 
time of study withdrawal or at the completion of the study. 
12 
 
13 
All females will be given a urine pregnancy test during the initial screening and the morning of or at least 24 
14 
hours prior to each DEXA scan. The test will be read by a female member of the study team and the results 
15 
will be shared with the volunteer. If the pregnancy test is positive, the volunteer will be excluded from 
16 
further participation in this study. 
17 
 
18 
All volunteers must be willing to consume only food and beverages provided by study staff during the 48-hr 
19 
normalization period and on glycogen depletion protocol days.  Additionally, volunteers must be willing to 
20 
refrain from any addition exercise during this period.   
21 
 
22 
       C1.4  Consent Process   
23 
 
24 
Informed consent documents will be provided to each prospective volunteer in writing, as well as in an oral 
25 
group presentation by the principal investigator or his designee.  The purpose of this study, procedures 
26 
involved, risks, and expectations of volunteers will be explained.  The principal investigator or designee will 
27 
answer all group and private questions.  Interested participants will sign the informed consent form prior to 
28 
undergoing initial screening for which they will have blood drawn for study-specific clearance.  If they meet 
29 
all the medical selection criteria after completing the screening health assessment, and attending the 
30 
consent information meeting, they will begin the pre-study assessments including measurements of VO2peak, 
31 
DEXA, and dietary and activity logs.  A copy of the informed consent will be provided to the volunteer with 
32 
the original kept for study documentation.  No study procedures will occur prior to the volunteer giving 
33 
informed consent.  Volunteers who have already consented will be informed of any new information or 
34 
changes to the protocol that may affect their willingness and ability to continue participation in the study 
35 
using an approved consent addendum.    
36 
 
37 
C1.4.1  Research involving subjects with cognitive impairment or who lack capacity to 
38 
provide informed consent: N/A 
39 
 
40 
                 C1.4.2  Research involving non-English speaking subjects: N/A 
41 
 
42 
C1.4.3  Research involving a waiver of the requirement to obtain informed consent OR 
43 
alteration of the elements of informed consent: N/A 
44 
 
45 
C1.4.4  Research involving a waiver of the requirement for investigator to obtain a signed 
46 
consent form: N/A 
47 
 
48 
                 C1.4.5  Waivers of assent or parental permission when the research involves children: N/A 
49 
 
50 
                 C1.4.6  Research involving data collection for the USAMRMC Volunteer Registry Database 
51 

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1 
It is the policy of USAMRMC that data sheets are to be completed on all volunteers participating in this 
2 
research for entry into the U.S. Army Medical Research and Materiel Command Volunteer Registry 
3 
Database.  The information to be entered into this confidential database includes name, address, social 
4 
security number, study name, and dates.  The intent of the database is twofold: first, to readily answer 
5 
questions concerning an individual’s participation in research sponsored by the USAMRMC; and second, to 
6 
ensure that the USAMRMC can exercise its obligation to ensure research volunteers are adequately 
7 
warned (duty to warn) of risks and to provide new information as it becomes available.  The information will 
8 
be stored at the USAMRMC for a minimum of 75 years. 
9 
 
10 
C2.  COMPENSATION FOR PARTICIPATION   
11 
 
12 
Military and civilian personnel will receive $35 for each successful blood draw.  There are 20 blood draws 
13 
during the two glycogen depletion protocol days (10 per protocol day).  Volunteers completing all 20 draws will 
14 
receive $700.  If a volunteer does not complete the entire study, they will be compensated for the number of 
15 
successful blood draws they did complete.  Volunteers will not be eligible for any other form of compensation 
16 
during this study.    
17 
 
18 
C3.  WITHDRAWAL FROM RESEARCH PARTICIPATION   
19 
 
20 
Participants will be allowed to withdraw at any time without penalty or loss of benefits to which they would 
21 
otherwise be entitled.  An investigator may stop an individual’s participation in the study if the participant is 
22 
unwilling or unable to complete study procedures.  An investigator or the medical monitor may also withdraw a 
23 
participant if the individual becomes ill or injured or it would not be in the participant’s best interest to continue.   
24 
 
25 
C4.  PRIVACY FOR SUBJECTS   
26 
 
27 
To protect the participant’s privacy, any of their research-related records will be labeled or “coded” with an 
28 
assigned research participant number that will not include their name or social security number. Dr. Lee M. 
29 
Margolis will keep the link between participant number and the participant’s research records in a locked 
30 
cabinet.  Any documents that will require the participant’s name, such as the consent form, will be kept in a 
31 
locked cabinet separate from any research documents that contain the participant’s ID number.  The principal 
32 
investigator is the only person who will be able to match the research participant number with any of their 
33 
personal identifying information.    
34 
 
35 
When the results of the research are published or discussed in conferences, no information will be included 
36 
that would reveal the participants identity to others.  If photographs, videos, or audio-tape recordings of you will 
37 
be used for educational purposes, your identity will be protected or disguised.  All identifiable or recognizable 
38 
information (e.g., names and faces) will be covered in any photographs unless volunteers agree to sign a 
39 
photo release form. 
40 
 
41 
C5.  CONFIDENTIALITY PROCEDURES FOR RESEARCH RECORDS, DATA, HUMAN BIOLOGICAL 
42 
SPECIMENS   
43 
 
44 
All data and medical information obtained will be considered privileged and held in confidence.  Study 
45 
volunteers will be assigned unique subject identification (ID) numbers that will not contain any personal 
46 
identifiers such as name, social security number, address, date of birth, zip code, etc.  This study subject ID 
47 
number will be used on all data collection instruments, to include questionnaires, data collection forms, 
48 
computer records, etc.  A number will be assigned as each volunteer is medically cleared for participation.  A 
49 
master list linking the volunteers’ names and ID numbers will be kept in a separate locked file in the principal 
50 
investigator's or project manager’s office, or kept in a computer file with password-protected access restricted 
51 

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to the principal investigator and project manager.  When the results of the research are published or discussed 
1 
in conferences, no information will be included that would reveal identity.    All samples will be stored using the 
2 
subject identification number.  Samples will be shipped for analysis to USACEHR and Metabolic Solutions.  
3 
Remaining samples will be stored at USARIEM in a -80ºC freezer in room 322 or 304.   The volunteers name 
4 
or other identifiable information will not be included on any data, data collection sheets, specimens, or other 
5 
research records.   
6 
 
7 
Only personnel assigned to the research study by the principal investigator will have access to the data.  Hard 
8 
copy data records will be stored for a minimum of three years from the time the study is completed.  Electronic 
9 
data records will be maintained for a period of at least five years after the study has been completed.   
10 
 
11 
C6.  RISKS OF HARM, MEASURES TO REDUCE THE RISKS OF HARM, AND BENEFITS OF 
12 
PARTICIPATION 
13 
 
14 
       C6.1 Risks of Harm   
15 
 
16 
Research Procedure Name:  DEXA Scan 
17 
Research Procedure Description:  Volunteer will lay face-up on the DEXA densitometer table in shorts, t-
18 
shirts, and stocking feet.  Volunteers will be asked to remain motionless for the 8-10 min scan. 
19 
Research-related Risks:  The DEXA scan is an X-ray and is considered to be a no greater than minimal 
20 
risk procedure.  The radiation dose of the whole-body DEXA scan is 0.1 mrem.  This dose is equivalent to 
21 
approximately 1/250 of normal annual background radiation, 1/9 of the radiation received in a transatlantic 
22 
flight, or 1/30 of the radiation received in a chest X-ray.     
23 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  A quality assurance check will be 
24 
completed on the DEXA each day prior to its use; the software will not allow the use of the DEXA 
25 
densitometer if the quality assurance check fails.  All females will be required to have a pregnancy test the 
26 
day before, or the day of testing.  If you are pregnant you will not be scanned, nor will you be able to 
27 
continue the study. 
28 
  
29 
Research Procedure Name:  Indwelling Catheters 
30 
Research Procedure Description:  A 18-20 g x 1.25 inch needle will be used to guide a catheter into the 
31 
antecubital vein of the volunteer.  The catheter will be attached to saline to keep the line patent for multiple 
32 
blood draws.     
33 
Research-related Risks:  The risks of blood sampling are small and usually limited to local bruising or 
34 
swelling.  Also sometimes volunteers feel faint or may faint, and in very rare cases experience a seizure 
35 
during or right after a blood draw.  If the volunteer has had problems with fainting during blood draws in 
36 
the past, they may be more prone to them during future procedures.  If the catheter, the tube that is left in 
37 
the arm after the needle is removed, becomes clogged at any time during the protocol, we will have to 
38 
replace this to continue blood sampling and therefore the study. This will require another needle to be 
39 
inserted into your arm.   
40 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Trained technicians will use sterile 
41 
techniques to place the catheter; however, in spite of being careful there is a chance that the site may 
42 
become infected.  Volunteers should not give blood for eight weeks before or after this study.   
43 
 
44 
Research Procedure Name:  Venipuncture 
45 
Research Procedure Description: A 21 g x 0.75 inch needle will be used for single blood draws of the 
46 
antecubital vein.   
47 
Research-related Risks:  Venipuncture is a routine clinical procedure the medical community commonly 
48 
uses to obtain blood samples.  The immediate complications may be slight pain during the entry of the 
49 
needle into the skin, possible dizziness, syncope, and in very rare cases experience a seizure.  Dizziness 
50 
or syncope constitutes no long-term harm, and immediate relief is achieved by having the subject put their 
51 

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head down between their knees or lie down.  Additionally, a hematoma may result from the venipuncture, 
1 
but this is more unsightly than risk producing.  Late complications might include thrombosis of the vein due 
2 
to trauma or infection.  These complications are extremely rare. 
3 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Participant monitoring, aseptic 
4 
technique, including sterile disposable blood collection apparatus and adherence to standard medical 
5 
precautions reduce risk.  Trained technicians will perform all venipuncture. 
6 
 
7 
Research Procedure Name:  Lidocaine Injection 
8 
Research Procedure Description:  Roughly 8-10 mL of Lidocaine will be injected using a 25 g needle at 
9 
the site of the incision, superficially (i.e., skin) and within the vastus lateralis. 
10 
Research-related Risks:  Slight pain at the site of injection might occur. Although rare, anaphylactic 
11 
reactions may also occur following administration of lidocaine.  Unlikely, but possible side effects could 
12 
include: dizziness, confusion, shakiness, visual changes, nausea, and unusually slow heartbeat.   
13 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Volunteers will be instructed to Notify 
14 
study coordinator or PI immediately if an allergic (i.e., swelling, itching, rash, hives, difficulty swallowing, or 
15 
difficulty breathing) reaction occurs. In the case of severe reaction, lidocaine use will be discontinued.                 
16 
 
17 
        Research Procedure Name:  Muscle Biopsy 
18 
Research Procedure Description: A small incision will be made in the skin and fascia of the vastus 
19 
lateralis.  A 5-mm Bergstrom biopsy needle will pass through these incisions with manual suction while the 
20 
participant is under local anesthesia (1% lidocaine) to collect muscle samples. 
21 
Research-related Risks:  Percutaneous needle muscle biopsies have been established as a non-routine, 
22 
but safe research procedure. Similar to blood draws, there is a risk that volunteers will feel faint or may 
23 
faint, and in very rare instances experience a seizure during or right after a muscle biopsy.  If the 
24 
participant has had problems with fainting during blood draws or muscle biopsies in the past, they may be 
25 
more prone to them during future procedures.  There is some risk of post-biopsy infection, which can be 
26 
minimized by employing correct sterile procedures and carefully instructing volunteers on care of the 
27 
wound.  Nerve damage is possible.  Moderate stiffness, hematoma and swelling around the biopsy site 
28 
may occur following the procedure, but this usually resolves itself within several days.  Some minimal 
29 
scarring will accompany healing of the incision and formation of a hypertrophic scar or keloid is possible.  
30 
Although this is a rare event in fair-skinned persons, the incidence of hypertrophic scarring or keloid 
31 
formation associated with healing of a primarily closed skin biopsy site (i.e., one which was closed with 
32 
sutures immediately afterward) is 5-10% in dark-skinned persons.  Complications of bleeding can be 
33 
reduced by applying direct pressure to the wound following the biopsy.  If symptoms should occur, they 
34 
usually do not interfere with normal walking or heavier exercise.  Volunteers with evidence of bleeding 
35 
diatheses or with local skin infection or irritation, or recent anticoagulant medication (including aspirin), will 
36 
not be used.     
37 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Volunteers will be instructed about 
38 
precautions against hematoma and infection.  They will be given a handout outlining instructions for 
39 
proper care of the incision site (Appendix I).  Dr. Margolis will perform the procedure with sterile 
40 
technique according the USARIEM Muscle Biopsy SOP and record notes for each biopsy (Appendix J).  
41 
The medical staff at USARIEM will follow-up with participants within 3 days post-biopsy to monitor for any 
42 
sign of infection, bleeding, or hematoma.  To minimize the likelihood of hypertrophic scarring or keloid 
43 
formation, biopsy wounds will be closed as promptly as feasible. 
44 
 
45 
Research Procedure Name:  Exercise 
46 
Research Procedure Description:  All exercise testing in this investigation will occur on a cycle ergometer, 
47 
cycling at various levels of intensity based on exercise protocol. 
48 
Research-related Risks:  Exercise is generally considered safe and beneficial for individuals without 
49 
cardiovascular disease. The U.S. prevalence of fatal events is approximately 1:100,000 to 1:300,000 in 
50 
competitive high school athletes and increases to 1:15,000 to 1:50,000 in athletes over the age of 35.  
51 

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Current civilian and military guidelines state that individuals less than 40 years of age who have no 
1 
symptoms of or known presence of heart disease or major coronary risk factors have a low risk for cardiac 
2 
complications during vigorous exercise.  All volunteers in this study fall into this low risk category.  Local 
3 
muscle discomfort and fatigue may occur in active muscles during and shortly after exercise.  Muscle 
4 
soreness, ranging in intensity from mild to severe, may persist for 1 to 7 days.  
5 
Measures to Minimize Risks of Harm: (Precautions, safeguards):  Studies have confirmed the safety of 
6 
maximal exercise testing, particularly among apparently healthy persons without significant cardiovascular 
7 
risk factors.  As a precaution, there will be at least one spotter during all cycle exercise sessions, and 
8 
heart rate will be monitored in real time during testing.  In addition, exercise monitors and test 
9 
administrators will be CPR-certified.   
10 
       
11 
C6.2  Incidental or Unexpected Findings   
12 
 
13 
Health problems identified during the screening process will be documented and a copy provided to the 
14 
volunteer. The volunteer will be encouraged to make an appointment with their primary care provider for a 
15 
full evaluation of the problem. Volunteers with evidence of any physical, mental, and/or medical conditions 
16 
that would make the proposed studies relatively more hazardous will be excluded. 
17 
 
18 
      C6.3  Potential Benefits   
19 
 
20 
There is no direct health or other benefits related to participation in this study 
21 
 
22 
C7.  DATA AND SAFETY MONITORING   
23 
 
24 
       C7.1 Monitoring 
25 
 
26 
The PI will, with the assistance of Associate Investigators, continuously evaluate recruitment, the informed 
27 
consent process, adverse events, and protocol adherence and deviations in order to identify unanticipated 
28 
problems or risks to the volunteers associated with the research. The PI will ensure that the number of 
29 
volunteers recruited for this study complies with the protocol.  The PI or AI will submit a monthly summary 
30 
of all adverse events to the Research Monitor to determine whether the number of adverse events is 
31 
excessive for the risks outlined in the research protocol.  The PI (or acting PI) and onsite physician or PA 
32 
will discuss “discontinuation criteria” for individual volunteers as the study progresses, based on their 
33 
observations of the volunteer during testing or non-testing periods. Every morning, volunteers will be asked 
34 
the following questions to evaluate their readiness to test. 
35 
 
36 
• 
How have you been feeling well since the last test in our laboratory (below average, average, above 
37 
average)? 
38 
• 
Do you have any pain or symptoms to report that may affect our testing today (e.g., sinus congestion, 
39 
fatigue, muscle soreness, fever, tooth pain)? 
40 
• 
Have you reported all food and beverages consumed in the last 24 h that were not provided to you by 
41 
study staff? 
42 
• 
What time did you fall asleep last night and awake this morning? 
43 
• 
Did you perform any exercise or physical activities outside of study activities in the last 24 h? 
44 
 
45 
       C7.2 Research Monitor (as applicable)   
46 
The research monitor for this study is CPT Steven Yoo.  This individual is an appropriate subject matter 
47 
expert not associated with the protocol. The research monitor shall, at a minimum, review all unanticipated 
48 
problems involving risk to subjects or others, serious adverse events and all subject deaths associated with 
49 
the protocol and provide an unbiased written report of the event.  Other responsibilities may be assigned by 
50 
the MRMC IRB as needed. 
51 

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1 
C8.  REPORTABLE EVENTS    
2 
 
3 
       C8.1  Expected adverse events 
4 
 
5 
An adverse event is defined as any untoward or unfavorable medical occurrence in a human research 
6 
participant, including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or 
7 
disease, temporally associated with the individual’s participation in the research, whether or not considered 
8 
related to the individual’s participation in the research. 
9 
 
10 
A Serious Adverse Event is defined as any adverse event temporally associated with the subject’s 
11 
participation in research that is fatal, life-threatening, permanently disabling, requires inpatient 
12 
hospitalization, or results in congenital anomalies/birth defect, overdose or cancer, or based on appropriate 
13 
medical judgment, may jeopardize the participant, or may require medical or surgical intervention to 
14 
prevent one of the above outcomes. 
15 
 
 
16 
All medical events that the USARIEM Office of Medical Support and Oversight (OMSO) evaluates will be 
17 
reported to the ORQC.  The PI will report all adverse events to the Research Monitor, if one was appointed 
18 
for the study.   
19 
 
20 
Expected adverse events which are not serious are reported to the IRB at the time of continuing review of 
21 
the protocol.   
22 
 
23 
       C8.2  Unexpected adverse events and unanticipated problems 
24 
 
25 
All unanticipated problems involving risk to subjects or others, and serious adverse events that are 
26 
unexpected and determined to be at least possibly or definitely related to study participation, will be 
27 
promptly reported within one working day by phone (508-233-6306/4811) or email (usarmy.natick.medcom-
28 
usariem.mbx.usariem-rqc@mail.mil) to the USARIEM ORQC and the Commander.  These events will also 
29 
be reported to the HQ USAMRMC Institutional Review Board within one working day by phone (301-619-
30 
6240), or by e-mail (usarmy.detrick.medcom-usamrmc.other.irb-office@mail.mil). 
31 
 
32 
The research monitor is required to review all unanticipated problems involving risk to volunteers or others, 
33 
serious adverse events and all volunteer deaths associated with the protocol and provide an unbiased 
34 
written report of the event.  At a minimum, the research monitor should comment on the outcomes of the 
35 
event or problem, and in the case of a serious adverse event or death, comment on the relationship to 
36 
participation in the study.  The research monitor should also indicate whether he or she concurs with the 
37 
details of the report provided by the study investigator.  Reports for events determined by either the 
38 
investigator or research monitor to be possibly or definitely related to participation and reports of events 
39 
resulting in death should be promptly forwarded to the MRMC IRB. 
40 
 
41 
       C8.3  Adverse device effects: N/A 
42 
  
43 
      C8.4  FDA-regulated research under IND and IDE: N/A 
44 
 
45 
 
46 
 
47 
SECTION D:  REFERENCES 
48 
References 
49 
 
50 
1. 
Ivy JL. Muscle glycogen synthesis before and after exercise. Sports Med 1991;11(1):6-19. 
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Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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Date:  23 May 2019 
 
 
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2. 
Spriet LL. New insights into the interaction of carbohydrate and fat metabolism during exercise. Sports Med 
1 
2014;44 Suppl 1:S87-96. doi: 10.1007/s40279-014-0154-1. 
2 
3. 
Costill DL, Bowers R, Branam G, Sparks K. Muscle glycogen utilization during prolonged exercise on successive 
3 
days. J Appl Physiol 1971;31(6):834-8. 
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4. 
Margolis LM, Rood J, Champagne C, Young AJ, Castellani JW. Energy balance and body composition during US 
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Army special forces training. Appl Physiol Nutr Metab 2013;38(4):396-400. doi: 10.1139/apnm-2012-0323. 
6 
5. 
Hawley JA, Burke LM, Phillips SM, Spriet LL. Nutritional modulation of training-induced skeletal muscle 
7 
adaptations. J Appl Physiol (1985) 2011;110(3):834-45. doi: 10.1152/japplphysiol.00949.2010. 
8 
6. 
Mul JD, Stanford KI, Hirshman MF, Goodyear LJ. Exercise and Regulation of Carbohydrate Metabolism. Prog Mol 
9 
Biol Transl Sci 2015;135:17-37. doi: 10.1016/bs.pmbts.2015.07.020. 
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7. 
Treebak JT, Glund S, Deshmukh A, Klein DK, Long YC, Jensen TE, Jorgensen SB, Viollet B, Andersson L, 
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Neumann D, et al. AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic 
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and regulatory subunits. Diabetes 2006;55(7):2051-8. doi: 10.2337/db06-0175. 
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8. 
Jorgensen SB, Treebak JT, Viollet B, Schjerling P, Vaulont S, Wojtaszewski JF, Richter EA. Role of AMPKalpha2 
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in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse 
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muscle. Am J Physiol Endocrinol Metab 2007;292(1):E331-9. doi: 10.1152/ajpendo.00243.2006. 
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9. 
Canto C, Auwerx J. PGC-1alpha, SIRT1 and AMPK, an energy sensing network that controls energy expenditure. 
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Curr Opin Lipidol 2009;20(2):98-105. doi: 10.1097/MOL.0b013e328328d0a4. 
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10. 
Scarpulla RC, Vega RB, Kelly DP. Transcriptional integration of mitochondrial biogenesis. Trends Endocrinol 
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Metab 2012;23(9):459-66. doi: 10.1016/j.tem.2012.06.006. 
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11. 
Wilson GJ, Layman DK, Moulton CJ, Norton LE, Anthony TG, Proud CG, Rupassara SI, Garlick PJ. Leucine or 
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carbohydrate supplementation reduces AMPK and eEF2 phosphorylation and extends postprandial muscle 
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protein synthesis in rats. Am J Physiol Endocrinol Metab 2011;301(6):E1236-42. doi: 
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10.1152/ajpendo.00242.2011. 
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12. 
White MF. The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem 
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1998;182(1-2):3-11. 
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13. 
Dumortier O, Hinault C, Van Obberghen E. MicroRNAs and metabolism crosstalk in energy homeostasis. Cell 
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Metab 2013;18(3):312-24. doi: 10.1016/j.cmet.2013.06.004. 
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14. 
Kong YW, Cannell IG, de Moor CH, Hill K, Garside PG, Hamilton TL, Meijer HA, Dobbyn HC, Stoneley M, Spriggs 
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KA, et al. The mechanism of micro-RNA-mediated translation repression is determined by the promoter of the 
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target gene. Proc Natl Acad Sci U S A 2008;105(26):8866-71. doi: 10.1073/pnas.0800650105. 
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15. 
Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology. Nature 2011;469(7330):336-42. 
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doi: 10.1038/nature09783. 
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16. 
Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature 
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2008;455(7209):64-71. doi: 10.1038/nature07242. 
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17. 
Wada S, Kato Y, Sawada S, Aizawa K, Park JH, Russell AP, Ushida T, Akimoto T. MicroRNA-23a has minimal 
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effect on endurance exercise-induced adaptation of mouse skeletal muscle. Pflugers Arch 2015;467(2):389-98. 
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doi: 10.1007/s00424-014-1517-z. 
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18. 
Rivas DA, Lessard SJ, Rice NP, Lustgarten MS, So K, Goodyear LJ, Parnell LD, Fielding RA. Diminished skeletal 
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muscle microRNA expression with aging is associated with attenuated muscle plasticity and inhibition of IGF-1 
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signaling. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 
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2014;28(9):4133-47. doi: 10.1096/fj.14-254490. 
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19. 
Godlewski J, Nowicki MO, Bronisz A, Nuovo G, Palatini J, De Lay M, Van Brocklyn J, Ostrowski MC, Chiocca EA, 
43 
Lawler SE. MicroRNA-451 regulates LKB1/AMPK signaling and allows adaptation to metabolic stress in glioma 
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cells. Mol Cell 2010;37(5):620-32. doi: 10.1016/j.molcel.2010.02.018. 
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20. 
Ansari KI, Ogawa D, Rooj AK, Lawler SE, Krichevsky AM, Johnson MD, Chiocca EA, Bronisz A, Godlewski J. 
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Glucose-based regulation of miR-451/AMPK signaling depends on the OCT1 transcription factor. Cell Rep 
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2015;11(6):902-9. doi: 10.1016/j.celrep.2015.04.016. 
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21. 
Davalos A, Goedeke L, Smibert P, Ramirez CM, Warrier NP, Andreo U, Cirera-Salinas D, Rayner K, Suresh U, 
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Pastor-Pareja JC, et al. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc 
50 
Natl Acad Sci U S A 2011;108(22):9232-7. doi: 10.1073/pnas.1102281108. 
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22. 
Ryu HS, Park SY, Ma D, Zhang J, Lee W. The induction of microRNA targeting IRS-1 is involved in the 
52 
development of insulin resistance under conditions of mitochondrial dysfunction in hepatocytes. PLoS One 
53 
2011;6(3):e17343. doi: 10.1371/journal.pone.0017343. 
54 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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Date:  23 May 2019 
 
 
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23. 
Safdar A, Abadi A, Akhtar M, Hettinga BP, Tarnopolsky MA. miRNA in the regulation of skeletal muscle 
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adaptation to acute endurance exercise in C57Bl/6J male mice. PLoS One 2009;4(5):e5610. doi: 
2 
10.1371/journal.pone.0005610. 
3 
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Russell AP, Wada S, Vergani L, Hock MB, Lamon S, Leger B, Ushida T, Cartoni R, Wadley GD, Hespel P, et al. 
4 
Disruption of skeletal muscle mitochondrial network genes and miRNAs in amyotrophic lateral sclerosis. 
5 
Neurobiol Dis 2013;49:107-17. doi: 10.1016/j.nbd.2012.08.015. 
6 
25. 
Yamamoto H, Morino K, Nishio Y, Ugi S, Yoshizaki T, Kashiwagi A, Maegawa H. MicroRNA-494 regulates 
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mitochondrial biogenesis in skeletal muscle through mitochondrial transcription factor A and Forkhead box j3. Am 
8 
J Physiol Endocrinol Metab 2012;303(12):E1419-27. doi: 10.1152/ajpendo.00097.2012. 
9 
26. 
Aoi W, Sakuma K. Does regulation of skeletal muscle function involve circulating microRNAs? Front Physiol 
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2014;5:39. doi: 10.3389/fphys.2014.00039. 
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27. 
Turchinovich A, Weiz L, Langheinz A, Burwinkel B. Characterization of extracellular circulating microRNA. Nucleic 
12 
Acids Res 2011;39(16):7223-33. doi: 10.1093/nar/gkr254. 
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28. 
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and 
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microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9(6):654-9. doi: 
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10.1038/ncb1596. 
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29. 
Alevizos I, Illei GG. MicroRNAs as biomarkers in rheumatic diseases. Nat Rev Rheumatol 2010;6(7):391-8. doi: 
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10.1038/nrrheum.2010.81. 
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30. 
Donaldson A, Natanek SA, Lewis A, Man WD, Hopkinson NS, Polkey MI, Kemp PR. Increased skeletal muscle-
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specific microRNA in the blood of patients with COPD. Thorax 2013;68(12):1140-9. doi: 10.1136/thoraxjnl-2012-
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203129. 
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31. 
Hu J, Kong M, Ye Y, Hong S, Cheng L, Jiang L. Serum miR-206 and other muscle-specific microRNAs as non-
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invasive biomarkers for Duchenne muscular dystrophy. J Neurochem 2014;129(5):877-83. doi: 
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10.1111/jnc.12662. 
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Ortega FJ, Mercader JM, Moreno-Navarrete JM, Rovira O, Guerra E, Esteve E, Xifra G, Martinez C, Ricart W, 
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Rieusset J, et al. Profiling of circulating microRNAs reveals common microRNAs linked to type 2 diabetes that 
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change with insulin sensitization. Diabetes Care 2014;37(5):1375-83. doi: 10.2337/dc13-1847. 
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33. 
Margolis LM, Lessard SJ, Ezzyat Y, Fielding RA, Rivas DA. Circulating MicroRNA Are Predictive of Aging and 
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Acute Adaptive Response to Resistance Exercise in Men. J Gerontol A Biol Sci Med Sci 2016. doi: 
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10.1093/gerona/glw243. 
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Baggish AL, Hale A, Weiner RB, Lewis GD, Systrom D, Wang F, Wang TJ, Chan SY. Dynamic regulation of 
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circulating microRNA during acute exhaustive exercise and sustained aerobic exercise training. J Physiol 
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2011;589(Pt 16):3983-94. doi: 10.1113/jphysiol.2011.213363. 
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35. 
Baggish AL, Park J, Min PK, Isaacs S, Parker BA, Thompson PD, Troyanos C, D'Hemecourt P, Dyer S, Thiel M, 
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et al. Rapid upregulation and clearance of distinct circulating microRNAs after prolonged aerobic exercise. J Appl 
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Physiol (1985) 2014;116(5):522-31. doi: 10.1152/japplphysiol.01141.2013. 
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36. 
Margolis LM, Murphy NE, Martini S, Gundersen Y, Castellani JW, Karl JP, Carrigan CT, Teien HK, Madslien EH, 
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Montain SJ, et al. Effects of Supplemental Energy on Protein Balance during 4-d Arctic Military Training. Med Sci 
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Sports Exerc 2016;48(8):1604-12. doi: 10.1249/MSS.0000000000000944. 
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37. 
Margolis LM, Murphy NE, Martini S, Spitz MG, Thrane I, McGraw SM, Blatny JM, Castellani JW, Rood JC, Young 
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AJ, et al. Effects of winter military training on energy balance, whole-body protein balance, muscle damage, 
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soreness, and physical performance. Appl Physiol Nutr Metab 2014;39(12):1395-401. doi: 10.1139/apnm-2014-
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McInerney P, Lessard SJ, Burke LM, Coffey VG, Lo Giudice SL, Southgate RJ, Hawley JA. Failure to repeatedly 
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supercompensate muscle glycogen stores in highly trained men. Med Sci Sports Exerc 2005;37(3):404-11. 
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Fyfe CL, Stewart J, Murison SD, Jackson DM, Rance K, Speakman JR, Horgan GW, Johnstone AM. Evaluating 
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energy intake measurement in free-living subjects: when to record and for how long? Public Health Nutr 
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2010;13(2):172-80. doi: 10.1017/S1368980009991443. 
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Athletic Performance. Med Sci Sports Exerc 2016;48(3):543-68. doi: 10.1249/MSS.0000000000000852. 
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Lohman TG, Harris M, Teixeira PJ, Weiss L. Assessing body composition and changes in body composition. 
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Another look at dual-energy X-ray absorptiometry. Ann N Y Acad Sci 2000;904:45-54. 
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responses to moderate endurance exercise in skeletal muscle. Int J Sport Nutr Exerc Metab 2010;20(4):282-90. 
56 

Protocol Title:  Influence of carbohydrate availability on skeletal muscle and circulating microRNA expression 
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44. 
Peronnet F, Massicotte D. Table of nonprotein respiratory quotient: an update. Can J Sport Sci 1991;16(1):23-9. 
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Pallikarakis N, Sphiris N, Lefebvre P. Influence of the bicarbonate pool and on the occurrence of 13CO2 in 
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exhaled air. Eur J Appl Physiol Occup Physiol 1991;63(3-4):179-83. 
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Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal 
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Marabita F, de Candia P, Torri A, Tegner J, Abrignani S, Rossi RL. Normalization of circulating microRNA 
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Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization 
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of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 
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Lessard SJ, Rivas DA, Alves-Wagner AB, Hirshman MF, Gallagher IJ, Constantin-Teodosiu D, Atkins R, 
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and reveals novel exercise-regulated signaling networks. Diabetes 2013;62(8):2717-27. doi: 10.2337/db13-0062. 
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Camera DM, Ong JN, Coffey VG, Hawley JA. Selective Modulation of MicroRNA Expression with Protein 
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Ingestion Following Concurrent Resistance and Endurance Exercise in Human Skeletal Muscle. Front Physiol 
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training. J Physiol 2013;591(18):4637-53. doi: 10.1113/jphysiol.2013.255695. 
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Margolis LML, S.J.; Ezzyat, Y.; Fielding, R.A.; Rivas, D.A. Circulating microRNA are predictive of aging and acute 
21 
adaptive response to resistance exercise in men. J Gerontol A Biol Sci Med Sci 2016;EPub Ahead of Print. 
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SECTION E:  ABBREVIATIONS AND ACRONYMS 
27 
 
28 
PGC-1α; peroxisome proliferator-activated receptor gamma coactivator 1-alpha, SIRT1; sirtuin 1, 
29 
ACC; acetyl-CoA carboxylase 1, AMPK; 5' adenosine monophosphate-activated protein kinase, PDK4; 
30 
pyruvate dehydrogenase kinase 4, IRS1; insulin receptor substrate 1, GLUT4; glucose transporter 4, 
31 
CHO; carbohydrate, CON; control, AKT; protein kinase B, ERR; estrogen related receptor, PPAR; 
32 
peroxisome proliferator-activated receptors, DEXA; dual energy x-ray absorptiometry  
33 
 
34 
 
35 
 
36 
 
37 
 
38 
 
39 
SECTION F: DoD PRIVACY RULE AND PROTECTED HEALTH INFORMATION (HIPAA) 
40 
 
41 
 
42 
 NA – institution is not a covered entity 
43 
 
44 
 NA – will not use or disclose protected health information 
45 
 
46 
 HIPAA authorization will be obtained  
47 
 
48 
 
An application for waiver/alteration of HIPAA authorization will be submitted   
49