Identifying Therapeutic Targets of Accelerated Sarcopenia
Phase: NA
Conditions: Sarcopenia, Diabetes Mellitus, Aging
Sponsor: The University of Texas Medical Branch, Galveston

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Bed Rest in Accelerated Sarcopenia Study 
#17-0064 
 
Elena Volpi, MD, PhD 
 
National Institutes of Health 
 
 
 
Version 15.1 
 
January 25, 2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Introduction and Purpose: 
 
Accelerated Sarcopenia. Sarcopenia is a complex geriatric syndrome characterized by the loss of skeletal 
muscle mass, strength and function with aging15. It is a major contributor to the cycle of frailty 38 and increases 
the risk of falls, physical dependence, disability and mortality in older adults15,68,82.  While the loss of muscle mass 
and function occurs gradually with healthy aging, diseases or other insults and injuries can accelerate sarcopenia 
and lead to catastrophic declines in mobility and independence74.  
Type 2 Diabetes Chronically Accelerates Sarcopenia. Chronic diseases are associated with acceleration of 
sarcopenia. Among these, Type 2 Diabetes Mellitus (T2DM) has been reported in longitudinal studies to hasten 
the loss of muscle mass and strength in older persons, even after adjustment for comorbidities, glucose control, 
disease length and other factors58,72-74. Insulin resistance may play a role in this process62. However, we do not 
know which specific mechanisms are involved in the acceleration of sarcopenia by T2DM.  
Bed Rest Inactivity Acutely Accelerates Sarcopenia. Hospitalization is a well-known risk factor of functional 
loss in older adults14. Profound inactivity is the common denominator of hospitalization in geriatric patients, 
irrespective of disease or condition that led to hospital admission36. Inactivity during the hospital stay is 
associated with poorer clinical outcomes such as the length of stay3,51. More in general, bed rest inactivity is a 
major contributor to muscle loss and functional decline in older persons47,60 (Preliminary Data). The deleterious 
effect of bed rest on muscle size and function is significant and occurs even in the absence of disease22,60. 
However, the exact mechanisms by which acute inactivity accelerates sarcopenia are not well understood. 
Knowledge Gap. Conditions that accelerate sarcopenia, such as T2DM or acute immobilization, increase the 
risk of disability and dependency in older adults. However, the fundamental mechanisms by which sarcopenia 
accelerators induce loss of muscle mass and function are unknown.  As a consequence of this knowledge gap, 
and in the absence of specific therapeutic targets, it is not possible to develop innovative and effective strategies 
for prevention and treatment of accelerated sarcopenia in older patients.  
Significance. The proposed research is expected to spur a vertical advancement in our knowledge of the 
mechanisms that underlie accelerated sarcopenia in older adults. This work is significant because it will lead to 
the identification of novel, specific targets for the development of evidence-based interventions to prevent and 
treat accelerated sarcopenia and maintain independence in older adults at high risk of physical dependence.  
Benefit. Mobility disability and physical dependence are major concerns for older adults, and important 
contributors to rising healthcare costs in the geriatric population53.  In the year 2010, 26.9% of the U.S. adults 65 
and older had diabetes11. A decline in muscle mass and muscle quality in this patient population is associated 
with reduced mobility90 and increased risk of functional disability91. Hospitalization and bed rest for any cause 
are major events that interact with chronic conditions, further reducing function and independence in older 
adults14,47. By identifying the fundamental mechanisms that accelerate sarcopenia in these disabling conditions, 
we will be able to develop novel treatments for maintenance of independence. Thus, our research has the 
potential to positively impact not only healthcare outcomes but also costs over the long term. 
 
2. Background: 
Low muscle mass is a basic component of sarcopenia15. The association between muscle mass and 
function is not linear in healthy older adults35. However, conditions that accelerate sarcopenia decrease muscle 
mass at the same rate as strength, or faster3. A major determinant of skeletal muscle mass is contractile protein 
content, which is regulated by the balance between protein synthesis and proteolysis99. Incorporation of dietary 
amino acids into newly synthesized proteins is a fundamental process for maintenance and growth of skeletal 
muscle, because it allows for replacement of the essential amino acids released by proteolysis and lost with 
oxidation or transport to other tissues92. The stimulatory effect of an amino acid/protein meal on skeletal muscle 
protein synthesis is large and rapid48,84, and driven by essential amino acids, particularly leucine, which directly 
stimulate muscle protein synthesis by activating translation initiation via the mTORC1 pathway18,5. To activate 
mTORC1 signaling and protein synthesis, amino acids must first enter the muscle cell via amino acid transport. 
Amino acid transport is an active process that is emerging as a key regulator of skeletal muscle protein 
homeostasis and anabolism55. Amino acid transport is controlled by several factors 1,55. We have reported that 
dietary amino acid intake27,96, insulin 86 and resistance exercise26 can independently activate amino acid 
transporter expression and function in humans, and this effect is associated with increased muscle protein 
synthesis 19.     

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There is growing evidence from our group43,75,87 and others80 that healthy aging per se induces a mild but 
significant reduction in the response of muscle proteins to anabolic stimulation. This phenomenon has been 
termed “anabolic resistance” and may account for the slow loss of muscle mass with advancing age. It is 
characterized by a reduced anabolic response to low amino acid doses17,56,57,84,94 and insulin43. This is a true 
“resistance” because the maximal anabolic response to high doses of amino acids83,93,96 or insulin43 is preserved 
in healthy older adults and comparable to that observed in younger individuals.  
Our preliminary data suggest that a global, underlying mechanism of accelerated sarcopenia is an 
increase in anabolic resistance associated with a reduction in amino acid transport. With this application we will 
examine how amino acid transport is impacted by conditions accelerating sarcopenia and identify factors that 
control it.     
In Specific Aim 1 we will determine if a well-known chronic accelerator of sarcopenia, T2DM, reduces the 
sensitivity of amino acid transport to increasing doses of exogenous amino acids. In Specific Aim 2 we propose 
that short-term bed rest inactivity will reduce the sensitivity of amino acid transport to exogenous amino acids 
and that T2DM will worsen this negative adaptation. Upstream regulatory pathways will be explored also in this 
aim to identify promising therapeutic targets for interventions. 
 
Innovation 
The research we propose is innovative, in our opinion, for at least three major reasons: 
1. Conceptual Innovation. Our proposed studies will explore for the first time the potential global role of 
amino acid transport in conditions that accelerate sarcopenia chronically and acutely. By using a well described 
and common model of accelerated sarcopenia, T2DM, this research may also rapidly yield practical innovation 
that could be rapidly translated in the clinical setting.     
2. Methodological Innovation. Innovation also stems from our novel integrative and interdisciplinary 
translational approach that combines state of the art and novel methodologies to determine amino acid 
transporter expression and function and their upstream regulation.  These methodologies include: a 3-pool model 
tracer methodology to measure amino acid trafficking across vascular and intracellular compartments20; 
molecular techniques to measure regulation of muscle protein synthesis and amino acid transport26,27; and near 
infrared spectroscopy (NIRS) to determine the role of endothelial function and microvascular muscle 
perfusion86,87.  Inactivity is a well-established means to induce muscle atrophy. Bed Rest inactivity also reduce 
amino acid transport and transporter expression and leads to anabolic resistance22. In this application, we will 
innovatively use Bed Rest as a tool to modify the response of amino acid transport and transporter expression 
with the purpose of testing our hypotheses and identifying new therapeutic targets.  
3. Discovery of Novel Molecular Targets. Our secondary outcomes will include exploration of the role 
of novel regulatory proteins in relation to the changes in amino acid transport associated with T2DM. The basic 
assumption is that chronic and acute sarcopenia accelerators may reduce amino acid transport with different 
mechanisms. Thus, we will determine the potential role of newly identified amino acid sensing pathways such 
as the Sestrins-GATOR1/2 and the lysosomal vATPase/SLC38A9/Regulator complex in the activation, or lack 
of thereof, of mTORC1 signaling and amino acid transporter expression and function.  
Our approach ― unique and substantially different from the status quo ― is expected to unravel the key 
underlying mechanisms responsible for accelerated sarcopenia due to T2DM. The knowledge generated by our 
studies is needed to develop future evidence-based clinical trials of interventions. 
 
 
 
3. Concise Summary of Project: 
Subjects. We will test our central hypothesis in community dwelling, independent older adults with non-
insulin dependent T2DM. Since data previously gathered on healthy subjects will serve as controls for T2DM 
subjects, who are expected to be overweight or obese, we will limit inclusion to subjects with BMI 23-35 kg/m2.  
Those who have a BMI ≥ 35 kg/m2 will be considered for inclusion on a case-to-case basis, depending on whether 
or not thigh adiposity would impair muscle biopsy. Morbidly obese, frail or exercise trained subjects will be 
excluded, as these conditions can significantly impact muscle protein metabolism13,79,89, confounding the results. 
Subjects will be weight stable, as active weight loss in obese older adults can temporarily improve the response 
of muscle protein synthesis to anabolic stimuli88.  The study will enroll 80 subjects who will be recruited from the 
UTMB Pepper Center Volunteer Registry, the Diabetes Clinic for Seniors directed by Dr. Volpi, the UTMB Stark 

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Diabetes Center (Dr. Belalcazar), referral from other UTMB physicians, and advertisement in local media.  To 
aid in subject retention and encourage study adherence, transportation to and from study visits via Lyft will be 
offered to subjects at no cost to them. We will use the UT System Lyft contract and desktop app, so that subjects 
will not need to own or use a private smartphone to benefit from this service. 
Screening:  After carefully explaining procedures and risks, informed consent will be obtained from all 
subjects.  Eligibility will be assessed with clinical history, physical exam, questionnaires, functional testing, DEXA 
scan and a number of laboratory tests including blood count, metabolic panel, uric acid, lipid profile, coagulation 
panel, hemoglobin A1c, Hepatitis  B and C, HIV test, urinalysis and urine toxicology drug screening. The subjects 
will arrive fasting for their screening visits for blood measures.  An OGTT will be performed, which may involve 
the use of U-13C glucose tracer to better characterize glucose metabolism. Habitual physical activity will be 
measured using both StepWatch Activity Monitors (SAM) and pedometers for 1 week during the screening 
process. Rescreening of subjects who did not meet inclusion criteria for reasons that may resolve over time as 
determined by the investigator is allowed (e.g. dehydration, acute infections, medications, etc.).  Subjects will be 
rescreened within 3 months and only 1 rescreen will be allowed per subject.  
 
 
4. Study Design. Subjects will undergo a two acute metabolic experiments, one prior to and one immediately 
following the completion of 5 days of Bed Rest, in which we will test the effects of 7 or 21 g of EAA in older 
healthy and T2DM patients. The study will last a total of 7 consecutive days, including the bed rest staying and 
the following rehabilitation. Note that the rehabilitation period could be prolonged according to the physical 
therapist evaluation. If this will be the case, the physical therapist will provide extra rehabilitation treatment as 
usually occur during hospital provider. 
 
1) Run-In: Subjects will report to the 
Institute for Translational Sciences Clinical 
Research Center (ITS-CRC) the day prior 
to the experiment, in this occasion we will: 
• 
Follow current institutional COVID-19 
testing policy. If positive the subject will be 
referred to own PCP. If negative we will: 
• 
Admit subjects to the ITS-CRC. 
• 
Draw safety labs including blood 
count, metabolic panel, coagulation panel, 
urinalysis. Additional labs may be ordered 
by the study clinical provider, as clinically 
appropriate, if changes in the medical 
history have occurred between screening 
and admission. 
• 
Provide standardized meals (see above). 
• 
Hold vasodilators, monitor blood pressure, and manage hypertension with other medications if necessary 
43,75,87. 
 
2) Metabolic Experiment. The experimental procedure is designed to measure skeletal muscle amino 
acid transporter expression and function, whole body and muscle amino acid kinetics, muscle protein synthesis 
and proteolysis is depicted in Figure1.  The subjects will be studied in the post-absorptive state, after an overnight 
stay in the ITS-CRC. After admission, subjects will be given dinner at approximately 18:00 and a snack at 
approximately 21:00, after which they will be allowed only water ad libitum. At ~06:30 we will measure body 
composition by DEXA. At approximately 07:00 we will insert: an antecubital forearm venous catheter for systemic 
infusions; a retrograde venous catheter in the opposite hand placed in a heating pad for arterialized blood draws. 
At approximately 0900, we will place if possible a 3 French/ 8 cm or 4 French/ 12 cm pediatric central catheter 
in the femoral vein for blood draws.  If not possible the femoral catheter will not be placed and the metabolic 
parameters that need a femoral venous blood sample will not be  measured. Subsequently, we will start a primed-
continuous infusion of L-[ring-13C6]-phenylalanine, L-[5,5,5-2H3]-leucine, and [1-13C]-glycine to measure muscle 
Bx 
x 
x 
x 
x 
Blood 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
US/NIRS 
x 
x 
x 
x 
L-[ring-13C6]Phenylalanine, L-[5,5,5,-2D3]Leucine, L-[1-13C]Glycine   
0                1
            2
       3
            4
       5
            6
       7
            8
      9     
EAA 
Basal (3hr) 
Treatment (3hr) 
Catheters 
Tracer Prime 
Stop Tracer 
Figure 1. Metabolic Study design with approximate timing. 

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protein turnover as well as the amino acid transport rates through LAT1 (Phe, Leu), SNAT2 (Gly), and PAT1 
(Gly)55,93.  To reach the intracellular space, amino acids have to first be transported across the endothelium and 
then across the muscle fiber membrane66. To reduce risks we will use arterialized blood samples, rather than 
arterial blood, to measure arterial amino acid concentrations and enrichments. This method has been previously 
validated for amino acid and other metabolite concentrations8, and we have preliminary data indicating that it is 
also valid for amino acid enrichments. Whenever possible we will also perform a Flow Mediated Dilation test to 
detect endothelial function using Doppler Ultrasound and Near Infrared Spectroscopy. After an approximately 4-
hr baseline period, subjects will ingest the assigned amino acid mixture, as a bolus.  The mixture will contain 
EAA in the pattern found in animal protein22 and will be enriched with L-[ring-13C6]-phenylalanine, L-[5,5,5-2H3]-
leucine to maintain the isotopic steady state22 (labeled glycine will not be added because it is not included in the 
oral mixture).  Large vessel blood flow will be measured by Doppler86,87.  Microvascular nutritive flow is emerging 
as a potential regulator of muscle amino acid and protein metabolism85-87. Thus, we will measure it in the basal 
state (see above) and after amino acid ingestion using Near Infrared Spectroscopy (NIRS) and measuring blood 
flow using Doppler Ultrasound.  Frequent blood samples will be collected during the last hour of the basal period 
and the first and third hour of the amino acid period to measure amino acid concentrations and enrichments, 
glucose, insulin, cytokines and endothelin-1. T2DM is characterized by hyperglycemia, hyperinsulinemia, 
inflammation and endothelial dysfunction, which may have an impact on muscle protein metabolism 69,86. 
Additional serum samples will also be collected and stored for future analyses (e.g. other hormonal assays, 
proteomics, metabolomics).  Two muscle biopsies will be taken in each period to measure all pertinent amino 
acid and protein kinetic parameters, cross-sectional area, cell signaling, novel regulatory pathways, and gene 
expression (see Analytical Methods).  A portion of the biopsies will be collected for immunohistochemistry. A 
third incision may be made should the need arise. At the end of the experiment catheters will be removed and 
subjects will be given a meal. The metabolic study will take approximately 8 hours. 
 
3) Physical Inactivity (Bed Rest).  After the Metabolic Experiment is completed, subjects will start bed 
rest inactivity. UTMB has a long tradition of safely performing bed rest studies of any length (from a few days to 
up to 90 days) in adults of all ages, including seniors (see for example refs.22,31,32,70,77). The UTMB ITS-CRC is 
equipped with specialized rooms for bed rest studies, which include CCTV cameras for continual monitoring and 
recording of subject to ensure that bed rest is effectively maintained throughout the experimental period. 
Recordings may be retained for a period not to exceed 45 days for review and will then be erased. Highly trained 
nursing staff and other patient care personnel is available at all times. The following standard procedures will be 
implemented for subject safety and comfort: 
 
 
 
 
 
• 
A slight bed-back elevation will be permitted 
and subjects may raise their shoulders with 
two pillows.  
• 
Periodic change of position in bed to 
alleviate positional discomfort and to eat. 
• 
Serial compression devices (SCDs) and 
compression stockings will be worn to 
decrease DVT risk. 
• 
Low molecular weight heparin (day 1-4) 
• 
Standard of care in-bed physical therapy for 
bedridden patients involving passive range 
of motion of the lower extremity major joints. 
• 
In-bed bathing, hygiene activities and 
diuresis.   
• 
Bedside commode allowed for bowel 
movements. 
 
The total energy content of the subjects’ diet during bed rest will be reduced to account for inactivity. 
However, the protein content will remain at the pre-bed rest RDA level (0.8 g/kg/day)32,37. T2DM patients 
will continue their anti-hyperglycemic treatment with glinides and/or short acting insulin, adjusted as 
necessary.  Blood glucose will be continuously monitored during bed rest using a device called 
Continuous Glucose Monitor (CGM).  
 
4) End of Bed Rest. On day 5 of bed rest body composition by DEXA, Metabolic Experiment and 
functional tests will be repeated. After the tests, subjects will be allowed to de-ambulate with staff help. 

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5) Inpatient Rehabilitation.  Our pilot data indicate that three days of traditional rehabilitation is sufficient 
to restore physical function (measured by SPPB score) to pre bed rest levels in older subjects (see 
Figure 12 in Human Subjects). All subjects will be screened for glucose tolerance and postural 
hypotension prior to the resumption of weight-bearing activities. On day 1 they will focus on gradual re-
mobilization with mild stretching, walking, and cycle ergometry. Days 2 will consist of graded aerobic 
exercise with 30 min of treadmill walking at a moderate rate of perceived exertion and resistance 
exercise focusing on the lower body, core stability and postural control. Rehabilitation will be delivered 
by a trained staff member and overseen by Dr. Steven Fisher, PT, PhD.,  who will discharge you (based 
on his evaluation of your progression) with prescription for a home exercise program to maintain your 
functional level. During this period you will remain in the CRC and continue to receive prepared meals.  
Dietary energy intake will be increased to account for the higher physical activity level. 
 
 
5. Sub-Study Procedures: 
 
Specimens obtained during the study will be stored in the principal investigator’s laboratory and 
may be used in future research. For example, from time to time, new laboratory tests and procedures 
are developed that could use better understanding of the human body during metabolism studies.   
 
The extra muscle tissue/blood left over after the initial research study tests are done will be stored and 
used in future research. Samples will be kept for a maximum of 10 years and then destroyed. The 
samples will be used to look at muscle growth related pathways.  DNA will not be stored and no genetic 
testing will be done. Any future research will not involve additional specimens being taken or require 
any extra involvement from subjects. We will not need to contact the subjects again. The specimens 
will be labeled with a code number and will not directly link any of the subjects who partake in the study. 
 
 
6. Criteria for Inclusion of Subjects: 
• 
Age 60-85 yr 
• 
Body mass index: 23-35 kg/m2 
• 
Or BMI>35 if thigh adiposity does not impair muscle biopsy 
• 
Score ≥ 26 on the 30-item Mini Mental State Examination 
• 
Stable body weight for at least 3 months 
• Normal, non-diabetic (normal response to a 2-hr 75 g oral glucose tolerance test per American 
Diabetes Association criteria)4 
OR 
• 
T2DM  
 
7. Criteria for Exclusion of Subjects: 
• 
Insulin therapy that cannot be converted to non-insulin therapies, significant diabetic 
complications, or A1c > 8%. Those excluded based on A1c will be eligible for re-evaluation. 
• 
Impairment in Activities of Daily Living 
• 
≥ 2 falls/year or weight loss ≥ 10% in the past year 
• 
Significant Cardiac abnormalities considered exclusionary by the study physician (e.g., , CAD,)  
• 
Vascular disease  
• 
Use of anticoagulant therapy. (e.g., warfarin, heparin, Pradaxa) 
• 
Uncontrolled blood pressure: systolic >160 mmHg or diastolic > 100 mmHg 
• 
History of stroke with motor disability 
• 
Significant Chronic kidney disease 
• 
Anemia: Those excluded for anemia may be counseled and rescreened (CBC) in 30 days 
• 
Liver disease (AST/ALT 2.5 times above the normal limit) 
• 
Significant Respiratory disease (acute upper respiratory infection, chronic lung disease) 

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• 
Active cancer 
• 
History of mastectomy with axillary lymph node dissection  
• 
Recent (within 3 months), chronic treatment with anabolic steroids, systemic corticosteroids or 
estrogen. 
• 
HIV or active hepatitis B or C 
• 
Alcohol (CAGE questionnaire) or drug abuse (urine screening) 
• 
Any other condition or event considered exclusionary by the PI and faculty physician 
 
8. Sources of Research Material: 
 
Participants will provide verbal and written data specifically for research purposes. Study 
physician will have access to UTMB EPIC for subjects Measurement equipment (DEXA, stress test, 
food diary, timers for functional tests) will capture numerical and qualitative data. The SAM device will 
collect data on steps.  Only trained study staff will have access to written study data. These data will be 
confidential. 
 
 
9. Recruitment Methods and Consenting Process: 
 
Subjects will be recruited through an institutional review-board approved advertisement that will 
be posted at UTMB, local newspapers, and social centers as the library, recreation centers, university 
announcements, flyers, and by word of mouth.  Subjects will also be recruited by patient recruitment 
letters by mail, in-person at UTMB’s outpatient clinics, by physician referral, on-line recruitment using 
UTMB’s calendar, newsletters and websites, targeted Facebook and Twitter ads, and Trialfacts 
recruitment services. Referring providers sole responsibility is to refer potential subjects. Referring 
physicians will NOT be involved in recruitment, consenting or any other aspect of the study. They will 
only give us permission to contact any potential eligible patient in their care.   Subjects will be met at 
the Institute for Translational Sciences- Clinical Research Center.  Each subject will have a private 
room. The door will be closed during the consenting process. We will take all measures to ensure that 
subject privacy is maintained.  After responding to ads, subjects will be pre-screened and asked to 
come in for a screening visit at the ITS-CRC.  We will include all subjects who respond to our 
advertisement and invite them for a screening interview. If they qualify, they will be offered an 
opportunity to participate in the study. This will happen on a "first come first served" basis. We will study 
a normal healthy population between the ages of 60-85 representative of the ethnic makeup of the 
United States.  the details of the experimental procedures, including risks and benefits, will be explained 
by a research team member at the time of initial screening at the ITS-CRC. At this time, the subject will 
be given ample time to read the consent form, ask questions and discuss any concerns. After the subject 
has been satisfied, the subject will be given an opportunity to sign the consent form.  At every study 
encounter, each subject will be asked if they are still willing to participate in the study. Each subject will 
be asked if they have any questions, concerns, complaints, or comments. If the subjects have any of 
those listed, they will be appropriately addressed by the PI.   
 
Lyft service will be provided to those subjects that don’t have a car and need to reach the ITS-CRC site 
for screening and studies. 
 
10. Potential Risks: 
 
 
Possible risks and discomforts a subject might be exposed to during the course of the study include: 
 
Positive screening tests: One or more of the screening tests might be positive of for a medical condition. 
This information might be upsetting for the subject. The PI and study physician, Dr. Elena Volpi or our 
research team nurse practitioner will provide counselling to the subject in the event that a screening 
test is positive for a medical condition or a blood level is abnormal. The likelihood of this is low. 
 
Fasting: Risk of fasting is hypoglycemia, dizziness, nausea, and light headedness. Risk is low. 

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Blood draws: The subjects will undergo blood draws during the course of the study. The total amount 
of blood drawn will be approximately 170 ml for each of the two metabolic study days. The risks are 
pain, bruising and infection. People with borderline anemia might become more anemic with the 
repeated blood draws. Fainting is also a risk as some people faint at the sight of needles and/or blood. 
The risk of this event is low. 
 
COVID-19 rapid test: It involves a nasal swab, which may be uncomfortable and rarely cause a nose 
bleed. Risk is low. 
 
Anemia: Repeated blood draws or excessive sampling volume increases the risk of anemia. This is 
unlikely as the blood collected is less than a blood donation. 
  
Muscle biopsies: The risks of this procedure are pain, bleeding, bruising, infection, and a little scar at 
the site of the incision. The risks are low. The biopsy scar is in the shape and size of this "_" Some 
people may also faint at the sight of needles and/or blood. Up to 50% of subjects experience some 
soreness at the site of the biopsy up to 48 hours after it is done.  
 
 
Stable Isotopes Infusion: The only possible risk is sepsis (blood infection) and fever, but the risk is so 
small that the precise number is unknown. The isotopes are tested in order to assure their sterility 
(absence of microorganisms, such as bacteria, that may cause infections and fever). Antibiotics can 
treat these infections should they occur.  
 
DEXA Scan: Each DEXA scan will expose the subject to a small amount of radiation. Exposure to 
radiation can increase the risk of cancer and birth defects. The amount of radiation that a subject will 
be exposed to is so small that the exact risk is unknown. 
 
Femoral Venous Catheter: We will insert only a pediatric catheter in the femoral vein. The risks are the 
following: (A) Trauma to structures adjacent to the targeted vein (hematoma, nerve injury) is the most 
commonly recognized complication/error during the cannulation process (1.9-3.6% incidence during 
central venous cannulation are generally reported). Traumas rarely are serious, but the experience of 
the operator and the use of ultra-sound assistance can bring the risk to virtually 0%. (B) Complications 
related to the presence of the catheters, i.e., infection, thrombosis and thromboembolism. For patients 
with central venous catheters (CVC), an infection rate of 1.4 per 1,000 catheters is reported. However, 
most patients with CVC are immunosuppressed because of their underlying disease, therefore having 
a higher risk of infections than our research volunteers. Infection in the immediate post-insertion period 
(3-5 days) is commonly due to infection of the subcutaneous track caused by intraoperative 
contamination and as such are more easily diagnosed and treated than the feared systemic infections 
more prone to appear later. Catheter-related thrombosis occurs in 3.7-10% of patients. This 
complication is more likely to occur in patients with specific thromboembolic risk. Factors that contribute 
to thrombosis in patients are hypercoagulability (malignant tumors), venous irritation caused by 
chemotherapeutic 
agents 
and 
hyperalimentation 
fluids. 
 
The 
risk 
is 
moderate.  
 
Strength Testing: The subject will experience soreness and tenderness in the muscles for 2 to 3 days 
following exercise. This is normal and is one of the ways in which the human body responds to exercise 
in order for the muscle to grow. A cardiovascular event such as a heart attack is a very rare potential 
risk.  
 
Near Infrared Spectrometry (NIRS): No additional protection is necessary. The effect of heat due to 
near infrared light absorption is low – less than 0.5°C. 
 

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Deep Venous Thrombosis (DVT):  While there have been no reported DVT events associated with 
previous bed rest studies, there is a small risk of a DVT as a result of reduced physical activity or bed 
rest. We will use leg compression devices and low molecular weight heparin to further reduce this risk. 
 
Loss of Muscle Mass and Functional Capacity:  We expect that the subjects on bed rest will experience 
a loss of muscle mass and function during bed rest.  The magnitude of this loss has been previously 
described by our group22,60, and can be reversed by 3 days of intensive in-hospital rehabilitation (see 
preliminary data in Figure 12). 
 
Confidentiality: There is a general risk of disclosure of personal sensitive data in a clinical investigation. 
The risk is minimal. 
 
 
11. Subject Safety and Data Monitoring: 
Data Safety and Monitoring Plan. The ultimate responsibility for data and safety monitoring 
rests with Dr. Volpi.  The risk level associated with this study is estimated to be moderate. 
Our data safety and monitoring plan will include: 
1. Review during of screening results and any other available data at the weekly investigators’ 
meeting.  
2. A quarterly review of data safety and interim data analysis performed by the PI.  
3. An annual review performed by the UTMB IRB. 
4. Semi-annual patient safety review by a Medical Monitor, Mukaila Raji, MD, Chief of the Division of 
Geriatric Medicine, UTMB. 
Careful monitoring of the recruitment, enrollment, retention, adverse events, and study 
procedures will help protect the safety of study subjects, the quality of data, and the integrity of the 
study.  As part of the safety plan for this study, Dr. Volpi, who is ultimately responsible for the medical 
management of the subjects, will review each subject’s records to ensure that appropriate mechanisms 
to protect the safety of study participants are being followed; that protocol requirements are being 
adhered to; and that data is accurate, complete, and secure.  Subject records include consent forms, 
case report forms, flow of data forms, laboratory specimen records, inclusion/exclusion forms, adverse 
event logs, and medical charts.   
Non-serious, anticipated adverse events (AE) include hematoma, pain, infection, syncope, and 
loss of subject confidentiality.  If an anticipated adverse event occurs, Dr. Volpi will be immediately 
notified and a note will be entered into the subject's CRC chart.  Dr. Volpi will be responsible for 
evaluating each AE and determining attribution as well as the impact of the AE on the risk/benefit ratio.   
All unanticipated, serious, fatal and/or life-threatening adverse events will be reported to the IRB 
within 24h of occurrence.  The IRB and the PI will be responsible for determining whether modifications 
to the protocol and consent form are required.  If a determination is made that participants are found to 
be exposed to excessive risks in relation to anticipated benefits, the study will be immediately 
suspended.  Studies will not resume until modifications are made that are deemed to result in an 
acceptable risk/benefit ratio by the PI and the IRB.  Aggregate reports of adverse events will be prepared 
as mandated and forwarded to the IRB and ITS-CRC for review. 
Plan for Adverse Event Reporting. The PI will be responsible for evaluating each adverse 
event and determining attribution as well as the impact on the risk/benefit ratio, using the following 
Grading and Attribution criteria: 
Mild – transient laboratory test alterations that do not suggest injury; discomfort noted but no 
disruption of daily activities; no therapy, or only symptomatic therapy required. 
Moderate – Laboratory test alterations indicating injury without long-term risk, discomfort 
sufficient to modify normal daily activity, specific therapy required (i.e., more than symptomatic). 
Serious – laboratory tests indicating a serious health threat or permanent injury, incapacity, 
inability to work, or to perform normal daily activity, hospitalization required or prolonged, emergency 
treatment required, life-threatening events, death. 

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The attribution scale assesses the relation of the event to the study procedures. Dr. Volpi will 
judge whether an adverse event is: 1) not related; 2) 
possibly related; 3) probably related; or 4) definitely related 
to the study interventions. 
 
 
 
12. Procedures to Maintain Confidentiality: 
 
All study data will be collected in REDCap by the research team. Paper data flow sheets 
or case report forms will be stored in locked file cabinets in a secure area of the PI’s recruitment office.  
Interim data will be reviewed by the PI.  Any appearance of a Conflict of Interest by the research team 
will be avoided by the periodic monitoring by UTMB’s Conflict of Interest Committee.  The subjects’ right 
to confidentiality will be protected at all times and no subject will be identified by name in any publication 
that results from this research. 
 
 
13. Potential Benefits: 
 
The only potential benefits for the participation in this protocol are related to the knowledge of 
the results of the screening tests that will be released to the volunteer. The knowledge collected from 
the present studies may be of benefit to society, as these studies will help develop the scientific basis 
for designing treatments for reducing loss in muscle mass and function in older subjects with T2DM. 
 
Importance of the Knowledge to be Gained. These studies will allow us to identify the biological 
mechanisms by T2DM accelerate sarcopenia in older individuals.  Since T2DM is quite common in older 
adults and increase the risk of physical dependence, the knowledge to be gained will be very important 
to develop interventions to maintain independence in older adults. 
 
13. Statistical Analysis: 
 
Primary outcome variables will be measures of amino acid transport.  All other variables will be 
treated as secondary outcomes. We will first compute the basal-to-acute treatment change for each 
variable in each subject. Experiment to test Aim 1 is designed using a  2×2 factorial approach with no 
repeated measures (see Table 1).  A standard 2×2 ANOVA model will be used to test the main effects, 
treatment by group interactions, and contrasts of interest, particularly the interaction effect of EAA dose 
(Aim 1) on challenges to the metabolism by T2DM.   
 
 Experiment for Aim 2 is designed using a 2×2 factorial approach with repeated measures across time 
(see Table 2). We will then use a standard repeated measures 2×2 ANOVA model to test the main 
effects, treatment by group interactions, and contrasts of interest, particularly the interaction effect of 
amino acid dose (Aim 1)/exercise (Aim 2) on the effect of bed rest. For each variable we will also test if 
there are basal differences between groups using one way ANOVA. If basal differences are found, we 
will then use the basal values as covariates.  Factors such as age, sex, and BMI will also be considered 
as covariates. The same analytic plan will be used for secondary outcome variables, and the results 
will be used for the overall interpretation of our study.  To further elucidate potentially important 
mechanistic relationships between the primary and secondary outcomes, along with their relationship 
to demographic characteristics such as sex, we will also use multiple regression models and other 
techniques, such as principal components analysis. Differences will be considered significant at 𝑝<0.05. 
Post-hoc testing will be carried out using the Tukey-Kramer test. All calculations will be conducted in 
SAS (version 9.2 or later; SAS Institute Inc., Cary, NC).  As necessary, responses will be transformed 
to assure that model assumptions of normality and constant group variance are met.  Results will be 
presented as mean ± SD. 
 
Table 1.  Groups for Aim 1. Older adults with T2DM and
healthy controls will be randomized to two groups 
(n=8/group) receiving 7 g or 21 g of EAA.  
 
Essential Amino Acid Dose 
7 g 
21 g 
Control 
C7 
C21 
T2DM 
D7 
D21 

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Sample Size and Power Calculation. Sample size and power 
have been calculated for the primary endpoints for the 
planned ANOVA model (see Statistical Analysis). All 
calculations were conducted using our preliminary data and 
the nQuery software package. We have not powered the 
experiments to measure sex differences because in our 
experience, and under similar experimental conditions, 
muscle protein metabolism does not behave differently in 
women and men44, regardless of age64.  However, since 
there are no data on the effect of gender on skeletal muscle 
protein turnover and amino acid transport in bed rest and T2DM, we will balance groups by sex. In 
Table 3 we report the expected differences as original or log transformed units, the SD, the calculated 
n/group and the n/group including subjects to offset the expected attrition of 12%.  
 
 
Group Randomization. All recruited subjects who had signed the consent form before screening will be 
randomized to receive 7g or 21g of EAA to test Aim 1.  To contain experimental costs and improve 
efficiency, subjects in C7 and D7 will be the control group BR-C7 and BR-D7 in Aim 2. Subjects in BR-
C7 and BR-D7 will then be randomized to receive standard of care (BR-C7 and BR-D7) or intense 
physical therapy (BR-ExC7 and BR-ExD7) during Bed Rest.  
 
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BR-ExD7 

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