Proteomic investigation of human skeletal muscle before and after 70 days of head down bed rest with or without exercise and testosterone countermeasures

Introduction Long-term head-down bed rest (HDBR) results in musculoskeletal losses similar to those observed during long-term space flight. Agents such as testosterone, in addition to regular exercise, are effective countermeasures for reducing loss of skeletal muscle mass and function. Objective We investigated the skeletal muscle proteome of healthy men in response to long term HDBR alone (CON) and to HDBR with exercise (PEX) or exercise plus testosterone (TEX) countermeasures. Method Biopsies were performed on the vastus lateralis before (pre) HDBR and on HDBR days 32 (mid) and 64 (post). Extracted proteins from these skeletal muscle biopsies were subjected to 2-dimensional gel electrophoresis (2DE), stained for phosphoproteins (Pro-Q Diamond dye) and total proteins (Sypro Ruby dye). Proteins showing significant fold differences (t-test p ≤ 0.05) in abundance or phosphorylation state at mid or post were identified by mass spectroscopy (MS). Results From a total of 932 protein spots, 130 spots were identified as potentially altered in terms of total protein or phosphoprotein levels due to HDBR and/or countermeasures, and 59 unique molecules emerged from MS analysis. Top canonical pathways identified through IPA included calcium signaling, actin cytoskeleton signaling, integrin linked kinase (ILK) signaling, and epithelial adherens junction signaling. Data from the pre-HDBR proteome supported the potential for predicting physiological post-HDBR responses such as the individual’s potential for loss vs. maintenance of muscle mass and strength. Conclusions HDBR resulted in alterations to skeletal muscle abundances and phosphorylation of several structural and metabolic proteins. Inclusion of exercise alone or in combination with testosterone treatment modulated the proteomic responses towards cellular reorganization and hypertrophy, respectively. Finally, the baseline proteome may aid in the development of personalized countermeasures to mitigate health risks in astronauts as related to loss of muscle mass and function.

If YES, a signed letter on institutional letterhead must be submitted from the PI. The letter must answer affirmatively and disclose the relationship with the company or entity arrangements. A related financial interest does NOT automatically mean that the investigator cannot participate in the research. The CPHS will determine if the financial and or other conflict of interest can be reduced, eliminated or managed in order to allow participation in the research project.
If NO, a signed letter on institutional letterhead must be submitted from the PI. The letter must include the following statement:  Exposure to microgravity is known to result in a number of physiological impairments including loss of muscle mass and strength 1 loss of bone mass [2][3][4][5] , reduced erythropoiesis 2, 6-8 , reduced myogenic differentiation 9 , and a functional decline in the hypothalamic-pituitary-gonadal axis (HPGA) and circulating testosterone concentrations 10 . Current countermeasures have been largely ineffective at preventing or reversing the physiologic decrements that occur in response to spaceflight. While in-flight exercise is currently the most effective and feasible countermeasure against musculoskeletal losses, both isokinetic and isotonic exercise have been shown to further reduce endogenous testosterone concentrations in a simulated spaceflight model (i.e. bed rest) 11 . As mission-length is continuously increasing and the recurring visions of NASA including human exploration of Mars, optimization of implemented countermeasures is of high importance. As stated in the 2008 Evidence Report Summary: Risk of Impaired Performance Due to Reduced Muscle Mass, Strength, and Endurance: "Without doubt, transport between the Earth and Mars as well as the return trip represents the greatest risk to humans encountered in the history of human spaceflight." Our research team has extensive experience with the clinical use of androgens and amino acids in several populations including older and younger men and women. We have recent data on testosterone treatments in older men that extends 5 months (clinicaltrials.gov ID: NCT00957528) and are currently conducting an NCI funded clinical trial investigating the use of testosterone and amino acid treatments for 10 weeks in female cancer patients (NCT00878995). Further, our scientific and medical expertise at UTMB in conducting studies of muscle and bone metabolism make us uniquely qualified to undertake the proposed project. Finally, we are uniquely positioned to conduct the proposed research as our laboratory and study personnel are in close proximity to the NASA Flight Analogs Research Unit at UTMB.

2.0
Please upload any documents supporting the above background and significance statement.

ID Date Modified
There are no items to display 3.0 Preliminary Studies: Preliminary data often aid reviewers in assessing how valuable the project is likely to be. If graphs or tables are used to convey information, please maintain a consistent style.
a Testosterone as a countermeasure in Astronauts Testosterone promotes muscle protein synthesis. Testosterone increases muscle protein synthesis through its interaction with the androgen receptor and by enhancing muscle-specific IGF-I expression 12,13 . Testosterone has also been shown to increase satellite cell number and increase myogenic differentiation of pluripotent stem cells through the Wnt signaling pathway 14,15 . Furthermore, testosterone has been shown to reduce systemic inflammatory cytokines such as TNFa, IL-6 and IL-1b [16][17][18] , capable of mediating NF-κB dependent muscle proteolysis.
Rationale for maintaining physiological testosterone concentrations. The rationale for the use of testosterone as a countermeasure to maintain physiologic levels of testosterone is based on the following scientific evidence in men and women: 1) we have demonstrated that models of stress and trauma cause significant impairments in endogenous testosterone production and are associated with significant losses in muscle mass and strength 19 ; and 2) simulated microgravity (i.e. stress) and spaceflight are both capable of disrupting the hypothalamic pituitary gonadal axis (HPGA), leading to reduced endogenous production of testosterone. There are reports of lowered testosterone concentrations in male astronauts 20 , and animals 21-23 during real or simulated spaceflight. These observations during microgravity share striking similarities with the changes that take place during aging. Under terrestrial circumstances, the 46% drop in circulating testosterone noted in the astronauts studied by Strollo et al. would clinically classify them as hypogonadal 20 . Restoration or normalization of testosterone has been shown to ameliorate musculoskeletal losses in hind-limb suspended rats 24 and muscle mass but not strength in humans in the absence of exercise countermeasures 25 . However, no human studies have examined the therapeutic efficacy of testosterone in conjunction with an exercise protocol at preventing or reversing the many negative physiologic alterations that occur in response to bed rest or space flight.
Rationale for the exclusion of females. While testosterone is deemed safe for use in women in low doses, we instead propose to study the efficacy of a testosterone countermeasure in men in this proof-of-concept investigation. We contend that if the efficacy and safety of testosterone can be shown under these conditions in male representatives of the astronaut population, similar treatment paradigms can be tested in female representatives in future protocols.
Rationale for using testosterone in a cyclic dosing paradigm. For men with suppressed levels of endogenous testosterone, the standard of care treatment is an injection of intramuscular (IM) testosterone either weekly (100 mg IM) or every two weeks (200 mg IM). However, we have recent data demonstrating that a monthly cycled testosterone administration (i.e. dosed at 100 mg IM weekly on alternating months; one month on and one month off) is similarly anabolic and functional promoting to skeletal muscle as the continuous, standard of care approach, yet requiring only half the dose. Furthermore, we also have data showing that if we cycle testosterone weekly (100 mg IM weekly on alternating weeks; one week on and one week off), we can elicit a similar anabolic effect over 3-months, illustrating the potential versatility of alternative testosterone dosing regimens and the paradigm that similar positive anabolic results can be obtained with fewer doses of testosterone over time.
Our earlier research has shown that 6 months of continuous near-physiological testosterone administration increases LBM and muscle strength in healthy older men 26 . We have also shown that restoration of blood testosterone in severely burned male patients is anabolic to muscle protein, primarily by reducing muscle protein breakdown 27 . We recently published a study in which we administered a synthetic derivative of testosterone (oxandrolone, Oxandrin) to older women (7.5 mg po bid for 14 days) and measured mixed muscle fractional synthesis rate (FSR), anabolic markers androgen receptor and IGF-I protein expression at days 0, 5 and 14 28 . These promising results from various populations prompted us to investigate alternative cyclic dosing regimens, further improving the safety and efficacy of androgens for clinical use.
We recently completed a 5 month placebo controlled double blind study in older men where we compared the standard of care continuous dosing regimen to that of a monthly cycled administration in which older men were provided weekly testosterone for one month, alternated by one month of placebo. Our data show that continuous (100 mg/wk) or monthly cycled (100 mg/wk for the 1 st month, followed by one month placebo etc.) testosterone replacement increases muscle strength ( Figure 1) and LBM ( Figure 2) in older men with low normal endogenous testosterone concentrations, and reduces serum markers of bone resorption ( Figure 3). The androgen stimulation of skeletal muscle protein synthesis was not reduced during the "off" cycles for testosterone. Furthermore, our data show that monthly cycled testosterone regimens (one month on, one month off) have similar positive effects on muscle mass as continuous testosterone regimens. Additionally, these results indicate that a monthly cycled regimen leads to similar reductions in markers of bone resorption, without the associated decrease in markers of bone formation that follows after months of continuous administration ( Figure 4).
Here, we propose cycled testosterone replacement (two weeks of testosterone followed by two weeks off) offering a noninvasive, low-risk approach to prevent hormonal impairments that occur during long-term missions (i.e. 6 month Earth-Mars transit). A cycled testosterone approach offers several advantages over a continuous administration regimen: 1) The total amount of testosterone administered per person is greatly reduced, 2) It minimizes the potential side effects that may occur during continuous administration (elevated hematocrit, etc.), 3) It allows for a period of normalization for the HPGA during the "off' periods, and 4) It allows for repeated periods of increased protein anabolism during the "on" periods.

b. Project impact
Maintaining appropriate hormonal and nutritional balance during spaceflight is of critical importance in preventing musculoskeletal losses. It is our contention that the restoration of physiologic levels of testosterone is essential to prevent and/or restore spaceflight-and microgravity-induced losses in the musculoskeletal system. Current evidence suggests that the combination of testosterone, nutrition, and exercise will optimize the effectiveness of the existing exercise and nutritional countermeasures. Results from this proposal will lay the groundwork for the implementation of combinational countermeasures that will additively work to maintain preflight physiology of astronauts during long-term spaceflight missions.
c. Summary of Preliminary Data 1. Cycled testosterone treatment improves lean body mass (LBM), muscle strength, and bone metabolism.

2.
Cycled testosterone treatment appears to improve bone metabolism compared with continuous administration, with lower testosterone exposure over time. Aim 1: To determine the effect of cycled testosterone replacement in conjunction with resistance exercise during bed rest on muscle mass, muscle strength, and fatigue in men aged 24-55 years.
Aim 2: To determine the effect of cycled testosterone replacement in conjunction with resistance exercise during bed rest on markers of bone metabolism and bone mass in men aged 24-55 years.
2.0 *Hypothesis(es): List hypothesis(es) and an associated primary outcome measure(s) for each specific aim. If the study is not designated to test a hypothesis, describe the goals of the project as related to this specific aim. (ex: "determine hardware/logistic feasibility," "estimate mean and standard deviation of intervention effects," "evaluate degree of association between X and Y") Aim 1: Cycled testosterone replacement (weekly testosterone injections for 2 weeks, followed by 2 weeks off, etc.) in conjunction with exercise will have an additive effect in preventing loss of muscle mass and muscle strength in men representative of the astronaut population compared to exercise with placebo testosterone.
Aim 2: Cycled testosterone replacement (weekly testosterone injections for 2 weeks, followed by 2 weeks off, etc.) in conjunction with exercise will have an additive effect in preventing loss of bone mass and alterations in markers of bone metabolism in men representative of the astronaut population compared to exercise with placebo testosterone.

*Statistical Analysis:
Breifly describe what statistical analysis(es) will be applied in order to address the hypothesis/primary outcome.
Note: If the above hypothesis/primary outcome is quantitative, briefly descibe how qualitative data will be analyzed. Alternatively, if the purpose of the experiment is to estimate a characteristic of the response measure(s) (e.g. mean, SD of change, etc) then provide evidence that the proposed sample size will enable estimation within a reasonable degree of error.  Novel studies (first-time-ever) and descriptive/feasibility studies benefit from an understanding of the relationship between sample size and precision (may require assistance by a statistician).

Bed rest Control: 8 subjects
Bed rest + Exercise: 8 subjects Bed rest + Exercise + Testosterone: 8 subjects A power analysis was conducted to demonstrate that our sample size is sufficient to detect statistical differences and avoid type II errors. The sample sizes are based on the primary measures of metabolic and functional outcomes. We use conservative estimates of the variation where possible.
Primary metabolic measure: lean body mass: We recently demonstrated that a group of subjects on testosterone had increased LBM of 4.2 kg compared to controls that had a loss of 2 kg. The SDs were 1.6 and 2.2 26 . A sample size of 8 in each group will have 96% power to detect a difference in means of 4 assuming that the common standard deviation is 2 using a two group t-test with a 0.050 two-sided significance level. If the SD=2.5, there is still 84% power.
Primary metabolic measure: lean leg mass:Preliminary estimates for lean leg mass in males comes from Ferrando et al. in which treated subjects had increases of mean±SD of 4.2±1.6 and controls on average had losses of -2.0±2.2 26 . A sample size of 8 in each group will have 84% power to detect a difference in means of 4 assuming that the common SD is 2.5 using a two group t-test with a 0.05 two-sided significance level.
Primary functional measures: We recently demonstrated that a group of subjects on testosterone had increased leg extension, triceps extension, and bicep curl strength compared to controls who had no gain or losses 26 . Estimates of variation come from this work. A sample size of 8 per group will have 80% power to detect a difference in means of 15 kg between the groups for leg extension when the SD=10 using a two group t-test with a 0.05 two-sided significance level. With 8 per group there is 92% power for tricep extension to detect a difference of 10 kg (SD=5.5) and 98% power to detect a difference of 9 kg (SD=4) for bicep curl.
Summary of power for primary outcomes:We have demonstrated that with N=24 (N=8 for each of the treatment groups) should provide sufficient power to assess the specific aims and hypotheses for our study using calculations based on a two-group t-test. The power presented here is minimal as it does not take into consideration the repeated measures design which will benefit the power aspect of the study. In other words by using the proposed repeated measures mixed model, the power characteristics will be improved as the time points gather strength across time.

ID: Pro0028
View: 3.2 Explain Study design

-Explain Study Design
You indicated the following design characteristic: Study design characteristics selection: * Study design: Explain all selections listed above.
Male volunteers (24-55 years) will be randomly assigned to one of 3 bed rest groups (non-exercising control + placebo, exercise + placebo, exercise + testosterone) using a standard of care approach with NASA spaceflight nutritional requirements and standard exercise protocols as the control condition. An example of a 70-day bed rest timeline is provided in Figure 5.
Our study design is compatible with a head down tilt (HDT) bed rest protocol. It is important that the proposed testosterone countermeasure is tested under conditions that can be compared to other countermeasures.
Our general hypothesis is that the maintenance of normal physiologic male levels of testosterone during spaceflight will protect against the functional loss of muscle and bone, and will maximize the efficacy of existing resistance exercise protocols at preventing or reversing functional impairments that occur during bed rest.
To achieve these goals we will test the following specific hypothesis before, during and after 70 days of bed rest: Cycled testosterone replacement (weekly testosterone injections for 2 weeks, followed by 2 weeks off, etc.) in conjunction with exercise will have an additive effect in preventing loss of muscle mass, muscle strength, bone mass, and markers of bone metabolism in men representative of the astronaut population compared to exercise with placebo testosterone.
Specific Aim 1A: To determine the effect of cycled testosterone replacement in conjunction with resistance exercise during bed rest on muscle mass, muscle strength, and fatigue in men aged 24-55 years.
Specific Aim 1B: To determine the effect of cycled testosterone replacement in conjunction with resistance exercise during bed rest on markers of bone metabolism and bone mass in men aged 24-55 years.
Design Characteristics -treatment / intervention categories, other experimental variables (eg: time in bedrest) -total number of subjects Nested -separate subjects in each treatment category Testosterone replacement countermeasure: During the respective treatment periods, weekly placebo or testosterone enanthate injections (100 mg/wk) will be administered.When the nadir serum total testosterone concentration exceeds the normal range, we will reduce the next intramuscular injection from 100 mg to 75 mg and continue to monitor the nadir serum total testosterone concentration. If the nadir value persists above the normal range, we will reduce and additional 25 mg to 50 mg weekly.
Exercise countermeasure:Our protocol is compatible with and will utilize the Integrated Resistance and Aerobic Training (iRAT) protocol that is planned for testing in bed rest.
Nutrition: Our protocol is compatible with, and will utilize, the standard FAP dietary guidelines.
Blood Collection.We will collect morning fasting blood before iRAT, before bed rest, weekly during bed rest and after bed rest (12mL each) for measurements of hormones (i.e. Testosterone, Estradiol), markers of bone metabolism (i.e. NTX, Osteocalcin, BAP, sRANKL), plasma amino acids, inflammatory cytokines (Millipore Human Cytokine-13plex kit, Millipore, Billerica, MA), and other markers of protein catabolism including serum activin A and myostatin. Maximum blood required over the entire bed rest duration will be (13 x 12 mL) = 156 mL (see Blood Volume Request for requirement by test).
Urine Collection. Twenty-four hour (void by void) urine collections will be collected every week for measurements of hormones (Cortisol) and markers of bone degradation (Pyrilinks-D®).
Saliva Collection. Saliva will be collected every week for measurements of hormones (Testosterone, Cortisol) and other markers of protein catabolism.
Muscle Biopsies. We will collect muscle tissue from the vastus lateralis before, during, and after bed rest for the determination of proteins and RNA that regulate muscle protein synthesis and breakdown (mTOR,  Collected daily (post exercise, before lights-out): MD Anderson Brief Fatigue Inventory (BFI). Subjects will rate their fatigue on a 10-point scale (0= no fatigue, 10= as bad as you can imagine). Questions address fatigue; right now; average during past 24-hrs; worst during past 24 hrs; and level of interference with activities.
Collected 3-Times daily (on exercise days only): 10-Point Scale for Fatigue (10PSF). Identical to question #1 on the BFI. Subjects will indicate their fatigue on a 10 point scale (0= No fatigue, 10= Worst fatigue) before, immediately following, and 4 hours after completing the exercise regimen. (control subjects will be day/time matched to exercisers). This test may be completed orally, in which case staff will record the answer on the sheet.
Hardcopy questionnaires will be provided for subjects to fill out and return to FARU staff.
In addition, we have consulted with Dr. Kim Seeton and we will include the Buss-Durkee Hostility Index (BDHI) as an assessment of hostility as recommended. This will be administered weekly in conjunction with the POMS above.
Restoration of testosterone concentrations in hypogonadal men often results in improved mood (i.e. decreased depression, improved cognition) and decreased fatigue 29,30 . Therefore, the proposed low-dose testosterone treatment regimen is not expected to result in the negative mood changes often associated with high-dose misuse of anabolic steroids (i.e. irritability, aggression) 31 . Mood disorders are not prevalent among young males with low doses of chronic abuse (less than 300mg/week) 46 . The dose of testosterone proposed here (intermittent administration of 100mg/week) is well below this dose and an order of magnitude below the high doses where mood disorders are more likely to present (i.e. more than 1000mg per week) 46,47 . While the effects of testosterone replacement on mood changes during bed rest are yet unknown, no additional behavioral or psychological issues are anticipated to those normally encountered during long term bed rest 32 . If anything, we expect that testosterone treatment will result in improved mood and reduced fatigue. Male subjects will be randomized to one of three double-blinded groups (nonexercising control + placebo, exercise + placebo, exercise + testosterone). The pharmacy and safety monitor will have access to the coding key. The subjects, bedside nurses, PI and Co-Is will remain blinded throughout data collection and analyses. The attending physician, safety monitor, study lead, study management, and data manager may remain unblinded.
Randomized -intervention with 2+ groups -subjects are randomly assigned to a specific treatment / condition Double Blinded -subject and investigators do not know which condition subject is in In order to evaluated the efficacy of the proposed countermeasure this protocol will share measures from FTT, iRATS, and Standard Measures.
The primary dependent measures from this research will include muscle cross-sectional area and volume (determined by Magnetic Resonance Imaging (MRI)), lean body mass (LBM determined by intelligent Dual-energy X-ray Absorptiometry (iDXA)), fat mass (FM determined by iDXA), muscle performance measures (Biodex). The secondary dependent measures will include bone mineral density (BMD determined by iDXA), serum and urinary markers of bone turnover, markers of skeletal muscle signaling and inflammation, muscle fatigue, and quality of life.
Bone Mineral Density (BMD) using UTMB's new iDXA. Whole body iDXA scans will be obtained prior to bed rest, monthly during bed rest, and one month after bed rest. We will specifically measure LBM (total, trunk, leg), BMD (total, hip, spine), and FM (total, trunk, leg, %). UTMB's new iDXA is the latest state-of-the-art DXA that has the capability to measure muscle and bone with a much greater degree of sensitivity. Additionally, the new iDXA has the capability to measure additional parameters of fracture risk not obtained with the standard DXA.
MRI. To determine muscle cross-sectional area and total volume, subjects will have their lower body imaged in a clinical 1.5 Tesla magnet (GE Signa, Milwaukee, WI) located in the UTMB Department of Radiology. Briefly, image slices will be taken from the greater trochanter to the ankle joint with the muscle at rest. Image data files generated will be analyzed for appendicular total and muscle volumes using NIH Image software (NIH Image public domain software). Muscle volume (cm 3 ) will be computed as the addition of individual slice areas multiplied by slice thickness (5 mm). The full procedure including details of the anatomical positioning and MRI imaging techniques have been described previously (16).
Mood, Fatigue, and Quality of Life Questionnaires. Before, during and after bed rest we will measure mood and quality of life using validated questionnaires on a daily or weekly basis. Psychological state will be assessed weekly using the 65-item Profile of Mood States (POMS) questionnaire, a validated measure that yields an overall score (range 0-168, with higher scores indicating more disturbance in mood) and 6 subscales (tension-anxiety, depression-dejection, angerhostility, vigor-activity, fatigue-inertia, and confusion-bewilderment). To assess fatigue, the Multidimensional Fatigue Symptom Inventory (MFSI) 33 and the MD Anderson Brief Fatigue Inventory (BFI) 34 , will be completed every day. In addition, subjects will indicate their fatigue on a 10 point scale for fatigue (10PSF) (0= no fatigue, 10= as bad as you can imagine) before, immediately following, and 4 hours after completing the exercise regimen on exercise days only.
Muscle Biopsies.A total of three muscle biopsies (~100-200 mg) will be taken from the vastus lateralis, 10-15 cm above the knee 35,36 . One muscle biopsy will be taken prior to the start of bed rest, one during bed rest following the second cycle of testosterone and one at bed rest completion. Muscle biopsies will be taken using strict aseptic procedures from the lateral portion of the vastus lateralis muscle. This is a commonly performed procedure in our research studies 28, 36-38 (see Part V: Risks and Benefits). All tissue will be snap frozen in liquid nitrogen to stop all enzymatic reactions and frozen at -80˚C for later analysis of signaling, inflammatory markers, and catabolic markers such as those in the ActRIIB signaling pathway.
Skeletal Muscle Signaling. Muscle tissue from the biopsies will be used to assess cell signaling proteins. In the men we will examine key signaling proteins downstream to Testosterone: Follistatin, TCF4, Smad2/3, Smad7, MyoD,Myosin Heavy Chain 2 (MHC2), Androgen Receptor (AR), beta-Catenin, Myogenin, myostatin and other protein involved in the ActRIIB signaling pathway. A detailed description of the SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotting techniques is available in recent publications [39][40][41] .
Cardiac Compliance. Diastolic Function, measured noninvasively via transthoracic echocardiography, will be determined from the mitral inflow velocity (E-wave) and relaxation of the mitral annulus (e') before, during, and after bed rest. The ratio (or index) of E-wave/e' is independent of preload and can be used to assess cardiac compliance (ventricular stiffness). Normal diastolic function is represented by an E/e' < 8. An E/e' index of 8-12 suggests reduced compliance and and E/e' index > 12 suggests severe restriction. It is expected that inactivity results in increased ventricular stiffness. This measure is included to determine whether testosterone replacement in conjunction with exercise offers protection against possible bed rest induced changes in ventricular stiffness.
Oral Glucose Tolerance Test (OGTT). Glucose tolerance will be determine before, during, and after bed rest using a standard OGTT and a breath test. After an overnight fast (water allowed), an antecubital venous IV is placed for the collection of blood samples. During the baseline blood collection, a simultaneous breath sample is collected by having the subjects breathe into a breath collection bag fitted with one way valves. Following the baseline sample collection, a drink containing 75 g glucose and 150 mg U-13 C 6 -glucose is administered and consumed within 1 minute. From this point (t=0 minutes) blood and breath samples are collected at 30 min, 1hr, 2hrs, and 3hrs. For the collection of breath samples, the subjects are instructed to breath normally, hold their breath for 3 seconds, and exhale completely into the provided collection bags. Subjects remain at rest throughout the OGTT. The ratios of 13 CO2 to 12 CO2 in single breath samples are measured using a UBiT-IR300 infrared spectrophotometer (Otsuka Electronics Co., Ltd, Hirakata, Osaka, CV ≤ 1.0 %). All results are calculated as per mille (‰) change of 13 CO2 abundance from the baseline breath sample and expressed as ‰ delta over baseline (‰DOB). Insulin is an important regulator of skeletal muscle metabolism including glucose disposal as well as protein synthesis. It is anticipated that inactivity results in reduced glucose tolerance. This measure is included to determine whether testosterone replacement in conjunction with exercise offers protection against possible bed rest induced changes in glucose tolerance.
Muscle Function and Fatigue Testing. Before, during, and after bed rest the subject will perform muscle function testing on the Biodex dynamometer. Muscle function testing will include tests of maximal voluntary contraction (MVC), which involves producing the maximal amount of force one time, as well as skeletal muscle fatigue, which involves measuring the decline of contractile performance during repetitive contractions. These tests will be conducted using leg extension movements on a Biodex dynamometer. An integrated functional testing protocol has been developed in conjunction with Dr. Lori Ploutz-Snyder (see details of Dr. Ploutz-Snyder's functional testing protocol).

Vitamin D supplementation
Any overt metabolic or vitamin deficiency during a focused investigation involving intervention therapies should be regarded as a confounding factor. Of highlighted importance in this study is the possible presence of Vitamin D deficiency prior to enrollment because of its known importance in optimum bone health. While vitamin D deficiency is likely to affect muscle metabolism, little is known regarding the direct effects of vitamin D on skeletal muscle and about the presence of Vitamin D receptors in skeletal muscle 49 . However, the male reproductive tract is a direct target for vitamin D and while some have found associations between androgen concentrations and 25(OH)D concentrations in men 50 others have not 51 . Vitamin D supplementation (3,332 IU daily for 1 year) has recently been shown to benefit testosterone concentrations in overweight men (aged 20-49 years) with both vitamin D deficiency and low normal testosterone 52 . While significant and of high interest both clinically and scientifically, it should be noted that these increases were measured over a year and that, while vitamin D status improved markedly, testosterone concentration improved modestly (10.7 ± 3.9 nmol/l to 13.4 ± 4.7 nmol/l), staying well below the mid normal ranges (9.09-55.28 nmol/l) in these subjects . In order to minimize vitamin D status as a confounding factor and to ensure optimum health of the subjects before bed rest we will correct for vitamin D deficiencies in subjects prior to the onset of the study according to standard FAP procedures outlined in the exclusion criteria.
In addition we will utilize the following NASA Research results are available upon request by the subject.
Clinical data collected for safety monitoring will be included in the medical records.
Research data will not be included in the medical records. 2.0 * General description of study population: Healthy males aged 24-55 years.

* Indicate the inclusion and exclusion criteria for enrollment:
In addition to the standard FAP criteria we will follow the following exclusion criteria: i. Exclusionary medications will be an anticoagulant (Coumadin) because of the risk of bleeding during the biopsy procedure. Additional medications which will be disallowed for participation include: anabolic steroids, nitrates, antihistamines, andglucocorticoids.
ii. The subjects must have a minimal VO 2 max score of 30 ml/kg and minimal isokinetic knee extension strength/body weight ratio of 2.0 Nm/kg. iii. Subjects with LDL cholesterol above 200 mg/dL will be excluded because testosterone administration may elevate LDL cholesterol levels further. iv. Any man with a history of breast cancer or prostate cancer, or any indication of an occult carcinoma from an elevation of prostate specific antigen (PSA) above 3.0 ng/ml during screening by HTSF, or severe benign prostatic hypertrophy (BPH) by history (frequent urination, reduced stream) will be excluded. In this study we will give parenteral testosterone to young men in a cyclic fashion. We will check a screening PSA in these men and include men only with a PSA < 3.0 ng/ml. This is based on a recent study which showed that serum PSA levels of 4 or greater occurred at 2.1% in subjects in their 30's and 1.6% in subjects in their 40's 42 . The use of the PSA as a screen for prostate cancer is controversial. The American College of Preventive Medicine concluded in 2008 that there is insufficient evidence to recommend routine screening for prostate cancer with a PSA 43 . This position has not changed as recently reviewed although risk stratification of PSA-based screening is being considered 44 . Parenteral testosterone administration in men with erectile dysfunction increased PSA levels from baseline 37% but did not result in a doubling of the PSA level which is a standard measurement of disease progression in men being treated for prostate cancer 45 . Therefore, in our study we will check baseline PSA levels and monitor them monthly in our subjects receiving testosterone. PSA velocities greater than 0.75 ng x ml -1 x yr -1 while receiving testosterone will result in withdrawing the subject from the study and referral to an urologist. v. Subjects with liver dysfunction evidenced by a history of hepatitis B or hepatitis C, or by a three-fold elevation of liver enzymes (Alk phos, ALT, AST) above normal on screening will be excluded from the study. Testosterone can have hepatotoxic effects in some subjects and should be used with careful monitoring of LFTs. vi. Testosterone and other anabolic steroids can cause fluid retention that could worsen uncontrolled hypertension. Any subject with a blood pressure on three consecutive measurements taken at one week intervals that has a systolic pressure > 140 or a diastolic blood pressure > 90 will be excluded. This is the definition of hypertension as established by the Joint National Committee on Detection and Evaluation of High Blood Pressure. Subjects will be included if they are on two or less blood pressure medications and have a blood pressure below these criteria. vii. Any subject who has a major medical illness such as diabetes, chronic obstructive pulmonary disease, or sleep apnea will be excluded. Moreover, subjects will not have a recent history of smoking tobacco. Morbidly obese older men (BMI > 35) will also be excluded. viii. Subjects with evidence of kidney disease (serum creatinine > 2.0mg/dl) will be excluded from participation. ix. Any subject with thyroid disease as determined by an abnormal TSH level will be excluded from participation.
x. Any subject testing positive for HIV will be excluded as the effects of this disease on the inflammatory process are unknown. xi. Allergy to iodine, a component of Betadine which is used to prepare the subject's skin for invasive procedures, will be cause for exclusion from this study. xii. Subjects who engage in high intensity resistance training on a regular basis will be excluded. Many studies show that exercise, particularly resistance exercise increases muscle strength and muscle protein synthesis. xiii. Subjects with a known coagulation disorder or with clinical evidence indicative of a bleeding disorder (easy bruising, "free bleeders") will not be enrolled in this study due to potential problems that could arise from muscle biopsy procedures. xiv. Subjects with agitation/aggression disorder will be excluded. xv. Subjects with smoking history of >20 pack per year or unable to abstain from smoking for duration of the study will be excluded. xvi. Subjects with inplanted electronic devices (e.e pacemakers, electronic infusion pumps, stimulators) will be excluded. xvii. Subjects with recent (6 months) treated cancer other than basal cell carcinoma will be excluded. xviii. Subjects with a recent history of GI bleed (<12 months) will be excluded. xix. Subjects with hemoglobin or hematocrit lower than acceptable lab values will be excluded. xx. Subjects with a history of stroke with motor disability will be excluded. xxi. Subjects currently on a weight-loss diet will be excluded. xxii. Subjects with any other condition or event deemed exclusionary by the PI and physician will be excluded.
xxiii. Subjects will be screened with serum 25-hydroxyvitamin D level using 30-day lab values.
-If the 30-day value is <50 nmoles/L, the subject will be recommended to take 2000 IU/day of Vitamin D3 until admittance into the study at UTMB. Once admitted into the study, the subject will receive 2000 IU/day of Vitamin D3 during the pre-bed rest phase at UTMB.
-If the 30-day value is >50nmoles/L, the subject will not need to take the Vitamin D3 before admittance into the study at UTMB. The subject will also not need to receive the Vitamin D3 during the pre-bed rest phase of the study at UTMB.
-All subjects will receive 800 IU/day of Vitamin D3 during the in-bed rest phase of the study at UTMB. xxiv. Subject with HDL above or below the normal clinical ranges used by HTSF during screening will be excluded as this may be an indication of steroid use. xxv. Subjects with testosterone above or below the normal clinical ranges used by HTSF during screening will be excluded.
4.0 * If there are any age, ethnic, language, or gender-based exclusion criteria, please provide justification.
We will enroll only men in this protocol. If the efficacy and safety of testosterone in mitigating the musculoskeletal losses can be shown in men during these bed rest conditions then further considerations can be taken to include females in future protocols.

5.0
Are pregnant subjects excluded from participation?

Yes No
If yes, use the following statement "Female subjects in this study will model those in the astronaut population for whom participation in space missions is not allowed during pregnancy; therefore, for female subjects, a non-positive result from a pregnancy test will be required prior to inclusion in the study and prior to each experimental session." We anticipate having to recruit 32 subjects to reach our target number of 24 participants. We will utilize the inclusion and exclusion criteria described above. Risk discomfort Minimize risks View Testosterone: Androgen therapy in humans has been associated with increased fluid retention, an increase in acne and sex drive, changes in the blood cholesterol profile (higher total cholesterol and LDL cholesterol, lower HDL cholesterol), changes in hematocrit (increased red blood cell count), damage to the liver, sleep apnea, and the possibility that testosterone can be converted by the body into estrogen, leading to breasts enlargement and soreness. Medications like this one have been associated with potential prostate enlargement and with promoting the growth of cancer cells. Male subjects will be checked for this periodically during the study by means of a blood test called a PSA (Prostate-Specific Antigen) level.
Restoration of testosterone concentrations in hypogonadal men often results in improved mood (i.e. decreased depression, improved cognition) and decreased fatigue 29,30 . The proposed low-dose testosterone treatment regimen is not expected to result in the negative mood changes often associated with high-dose misuse of anabolic steroids (i.e. irritability, aggression) 31 . Mood disorders are not prevalent among young males with low doses of chronic abuse (less than 300mg/week) 46 . The dose of testosterone proposed here (intermittent administration of 100mg/week) is well below this dose and an order of magnitude below the doses where mood disorders are more likely to present (i.e. more than 1000mg per week) 46,47 . While the effects of testosterone replacement on mood changes during bed rest are yet unknown, no additional behavioral or psychological issues are anticipated to those normally encountered during long term bed rest 32 . If anything, we expect that testosterone treatment will result in improved mood and reduced fatigue.
Subjects will be monitored weekly for changes in hormone concentrations and presentation of symptoms. Subjects will be checked for this periodically during the study by means of a blood test called a PSA (Prostate-Specific Antigen) level.
View MRI: The risks associated with the MRI procedure are minor as it involves a static magnetic field and weak radio waves. These have not been known to cause health damage. This procedure, however, may cause episodes of claustrophobia as the subject will be required to enter an open ended well lit cylinder. The other risk associated with MRI's involves metals in and on the body. To lessen this risk subjects will be required to remove all metals including jewelry from their body. The noise generated by the MRI scanner can cause temporary hearing loss. Subjects will be provided with ear plugs. MRI scans will be performed by qualified MRI technicians in the UTMB Division of Radiology.
Subjects will remove all metallic objects before entering the MR environment or MR system room including hearing aids, beeper, cell phone, keys, eyeglasses, hair pins, barrettes, jewelry (including body piercing jewelry), watch, safety pins, paperclips, money clip, credit cards, bank cards, magnetic strip cards, coins, pens, pocket knife, nail clipper, steel-toed boots/shoes, and tools. Loose metallic objects are especially prohibited in the MR system room and MR environment. Subjects with metals within their body will be assessed by a study physician before they are allowed to proceed with the MRI procedure. Safety may be assessed using the following source: www.mrisafety.com.
Subject will be provided with earplugs to reduce noise and still allow communication during the MRI scan. View Anemia: Repeated blood draws or excessive sampling volume increases the risk of anemia.
We will draw <400 mL of blood during the entire protocol. View 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.
Long term immobility and dehydration increases the risk of Deep Vein 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. Subjects may move legs in supine position and are encouraged to maintain adequate hydration. Risk will be reduced in subjects randomized into one of the exercise groups. View Phlebotomy: The risks of phlebotomy include vasovagal reaction, pain and infection if aseptic procedure is not used.
The risks of phlebotomy include vasovagal reaction, pain and infection if aseptic procedure is not followed.
Aseptic procedures will be followed. View Exercise Testing: Risks may include muscle tightness, soreness, fatigue, inability to perform physical activity, and rarely a muscle strain or tear. In older individuals, there is a small risk of myocardial infarction during maximal exercise tests. This is generally assumed to be approximately 1 in 10,000.
Exercise will be supervised by trained personnel to prevent injury as per iRATS long duration bed rest protocol.
View Muscle Biopsies: Minor discomfort or pain, muscle soreness, bruising following muscle biopsy, minor bleeding during muscle biopsy, Infection, numb or tingling sensation lasting a period of days or months.
Drs. Sheffield-Moore, Paddon-Jones, Dillon, and Durham have together performed more than 2000 muscle biopsies with no serious side effects or complications, and have been certified by UTMB to perform such procedures. The vastus lateralis is regarded as a safe choice for biopsy sampling as there are no large vessels or major nerves in this region. The most common incidents associated with this procedure are bruising or black-and-blue marks in 1.4% of subjects, bleeding from the biopsy site in 0.2% of subjects, and pain lasting more than 3 days in 0.3% of subjects 48 . We will anesthetize the skin and subcutaneous tissue and make a 6-7 mm incision. A Trochar biopsy needle (Bergström) will be advanced through the fascia of the muscle with the cutting window closed. Then, with suction applied, the cutting cylinder will be opened and closed several times to collect tissue.
To minimize the risk of infection and bruising, an antibiotic ointment and pressure dressing are applied. Following the muscle biopsy, the skin will be closed with either sutures or liquid bonding agent and dressed with a compression bandage. Pain during tissue sampling will be minimized or eliminated with local anesthesia, while muscle soreness for the following 24-48 hours is easily controlled with overthe-counter strength ibuprofen or acetaminophen, and cold packs. The risk of infection is minimized by the use of aseptic technique and the application of antibiotic ointment. If infection is diagnosed, cultures will be taken and appropriate antibiotic therapy administered until resolution.
View iDXA: The risk involves low levels of radiation exposure (less than 0.3 mrem/whole body scan and less than 2.3 mrem including regional scans including AP Spine, Femurs, Calcaneus and Forearm for a person of average body size).
Subjects will be made aware of their exposure and risks prior to the scans and will have the choice to withdraw. If YES, please describe any potential for direct benefits to participants in this study No, there is no direct benefit to subjects in this study.

2.0
Describe any potential benefits to Society or Space Flight: Results from this study will further elucidate the role of testosterone in the maintenance of skeletal muscle and bone during long term bed rest as a model for space flight.
It is hypothesized that maintaining appropriate balance between hormonal status, nutritional status, and physical activity during spaceflight is of critical importance in preventing musculoskeletal losses. It is our contention that the restoration of physiologic levels of testosterone is essential to prevent and/or restore spaceflight-and microgravity-induced losses in the musculoskeletal system. Current evidence suggests that the combination of testosterone and exercise will optimize the effectiveness of the existing exercise and nutritional countermeasures. Privacy/Confidentiality (for Research Protocol) Subject privacy and data confidentiality will be maintained in accordance with 1) NASA Policy Directive (NPD) 7100.8, "Protection of Human Research Subjects"; 2) NASA Procedural Requirements (NPR) 7100.1, "Protection of Human Research Subjects"; and 3) to the extent allowed by Federal law. No 2.0 * How will the data for this study be collected and recorded?
All data collected will be de-identified and will include variables such as age, ethnicity, anthropometric measurements, and biological measurements (excluding genetic testing), MRI/iDXA scans, and strength/fatigue measures. Data from radiologic scans will be de-identified by UTMB information services (IS) personnel within the ITS-CRC before being released to the PI and CO-I's. Data will be stored electronically on access restricted servers or as hard-copies in locked cabinets within locked rooms at UTMB. Files will be password protected before sharing with non-UTMB bed rest. UTMB login credentials from authorized users 7.0 * Who, other than the specified study team, will have access to the study records or data? Specify their name, role, and affiliation.
By signing the subject consent form, subjects agree that data collected as part of the Flight Analogs Project may be shared, using only the number identifier, among appropriate investigators and collaborators of this study and their experiment personnel. All investigators, collaborators and experiment personnel in direct contact with research subjects will have completed the required Collaborative Institutional Training Initiative (CITI) online training.
The Life Sciences Data Archive (LSDA) will resume responsibility for archival of data according to a Data Submission Agreement to be developed with representatives of LSDA.
8.0 * How will the investigator maintain privacy in the research setting(s)?
All study data will be collected by the research team, reviewed by the PI, and stored in secure, locked files and/or databases in order to protect it from inadvertent loss or improper access. Data and other records will be identified by coded number only to maintain subject confidentiality. All the information obtained in connection with these studies will remain confidential as far as possible within state and federal law. Information gained from this study that can be linked to the subject's identity will not be released to anyone other than the investigators, the subject and the subject's physician. The results of these studies will be published in scientific journals without identifying the subjects by name.
9.0 * What are the consequences to participants of a loss of privacy (eg., risks to reputation, insurability, other social risks)?
Sense of insecurity due to public exposure, embarrassment, percieved loss of control of person, loss of trust by the subject toward the research team.
10.0 * If coded or identified data will be released, specify the persons/agencies to whom the information will be released. Please also indicate the provisions that will be taken to assure that the transmission of the data will maintain confidentiality: All study data will be collected by the research team, reviewed by the PI, and stored in secure, locked files and/or databases in order to protect it from inadvertent loss or improper access. Data and other records will be identified by coded number only to maintain subject confidentiality. All the information obtained in connection with these studies will remain confidential as far as possible within state and federal law. Information gained from this study that can be linked to the subject's identity will not be released to anyone other than the investigators, the subject and the subject's physician. The results of these studies will be published in scientific journals without identifying the subjects by name.
11.0 * When the study is completed and the data is submitted and accepted by NASA, please indicate your plans for the destruction of the local dataset.
Hardcopies of any data to be destroyed will be shredded. Electronic files containing study data will deleted from the UTMB network.

* Is this study collecting health information? Yes No
Describe any additional steps taken to assure that identities of subjects and any of their health information which is protected under the law is kept confidential. If photography, video or audio recordings will be made as part of the study, disposition of these recordings should be addressed here and in the consent form.
Subject privacy and data confidentiality will be maintained in accordance with 1) NASA Policy Directive (NPD) 7100.8, "Protection of Human Research Subjects"; 2) NASA Procedural Requirements (NPR) 7100.1, "Protection of Human Research Subjects"; and 3) to the extent allowed by Federal law. 3.0 Describe how, when, and where the consent process will be initiated: Standard FAP procedures will be used.

4.0
Who will obtain informed consent from subjects for this research?
Standard FAP procedures will be used. The complement PI and Co-I's will obtain consent.