NOTE: Received extension to 7/31/2009 per NSBRI (10/09)

This project represents a combination of two NASA-funded protocols, which investigated nutritional countermeasures to activity-induced sarcopenia. Our previous NASA project demonstrated that essential amino acid supplementation (EAA) throughout bed rest preserved lean body mass and reduced the loss of muscle function. However, the supplement we utilized was not feasible for delivery during space flight. The general goal of the current proposal is to reduce the amount of supplement required by optimizing the composition, the amount, and the timing of ingestion in order to effectively minimize the loss of lean body mass and function. Since the protocols of Drs. Wolfe and Ferrando were designed to be complementary, we have included them under one institutional review board (IRB) protocol at University of Arkansas for Medical Sciences (UAMS). Thus, this report will detail progress in both grant projects.

Since our move to UAMS, we have completed three important study groups related to the goals of this project. The aims we focused upon represented the testing of a more practical approach to the delivery of EAA in an astronaut population. We have completed the following specific aims:

1. Determine if the addition of 3 g of leucine to meals results in a stimulation of muscle protein synthesis over a 24 hr period. Two groups of subjects were studied during short-term bed rest; a control and a leucine group. In the control group, we enrolled 12 subjects and studied 8, while in the leucine group, we enrolled 8 and studied 8. While analyses are currently underway, preliminary data indicates that muscle protein synthesis was greater during the first day of bed rest with the leucine supplementation. It does not appear; however, that leucine supplementation was effective in stimulating muscle protein synthesis on subsequent days of inactivity.

2. Determine if ingestion of 7.5 g of EAA three times per day between meals will efficiently (gm protein synthesized/gm ingested) stimulate muscle protein synthesis. This group will be compared to a control group consisting of 2-day bed rest alone. We hypothesize that 7.5 g EAA will preserve muscle protein synthesis during bed rest. While 8 control subjects were studied, we also enrolled 10 and studied 7 subjects given 7.5 g of EAA TID (three times a day) throughout bed rest. Based upon our previous work, we anticipate that the addition of 7.5 g of EAA TID will result in an increase synthetic response. While these analyses are ongoing, this dosage allows us to ascertain a dose-response of EAA, given our previous results with the maximal response of 15 g.

Prolonged inactivity is inherent to trauma, serious injury, or major surgery. These events represent a significant stress to the patient such that the resultant muscle loss and weakness impairs subsequent rehabilitation. Studies reveal that the requirement for hospital intensive care often entails hypocaloric intake in the patient, thus further exacerbating the deleterious results of stress. This project is designed to investigate countermeasures which will maintain muscle mass during periods of prolonged inactivity, hypocaloric intake and stress. The proposed interventions are primarily nutritional, and are of unique design and composition so as to have a maximal benefit on a gram per gram basis. Thus, these investigations will be directly applicable and translatable to a patient populations.

A. Major Accomplishments

Space flight is a unique environment to the human physiology, and as such, induces changes in the hormonal environment that exacerbate the loss of lean mass. We hypothesized that muscular inactivity, when coupled with hypercortisolemia indicative of a stress response would result in a greater loss of muscle mass and strength. Since astronauts habitually consume a hypocaloric diet in space, we recently extended our investigations to include the combined effects of inactivity, stress, and hypocaloric feeding on muscle protein synthesis, mass, and strength. In a follow-up study, 2 groups of subjects were studied before and after 14d of bed rest. One group was subjected to the conditions of bed rest, hypercortisolemia, and hyporcaloric (80%) intake. The second group was subjected to the same conditions; however, 15g of an EAA supplement was given 3 times per day. Preliminary findings indicate that the loss of muscle mass and strength is exacerbated under these combined conditions of hypocaloric intake and hypercortisolemia. The addition of the EAA supplement appears to ameliorate these losses. Analyses are ongoing at this time.

Since our move to UAMS, we have initiated further studies to investigate the addition of leucine alone to meals in order to stimulate protein synthesis over a 24 hr period, and to investigate the interaction of EAA with a minimal amount of resistive exercise. To date, we have enrolled 7 subjects and completed study of 4. Preliminary data is not yet available. Since the protocols of Drs. Wolfe and Ferrando were designed to be complementary, we have included them under one IRB protocol at UAMS. Thus, this report will detail progress in both grant projects.

Our recent findings are consistent with our previous data and others (Stein) during space flight and indicate that muscular inactivity and stress (hypercortisolemia) represent strong catabolic stimuli which exacerbate losses in muscle mass and strength. This catabolism is further aggravated by the hypocaloric intake that is common during space flight. The implication for space flight is that sufficient anabolic stimuli, whether by nutritional, pharmacological, exercise, or combined means, are required to offset the reduction in muscle protein synthesis to maintain muscle mass and function.

The normal anabolic effect of meals was not be affected by prior ingestion of an amino acid and carbohydrate supplement.

The post-absorptive nadir in net muscle protein synthesis was no greater in subjects receiving supplementation than in control subjects.

In response to these findings, we investigated the effects of 28d of hypercortisolemia alone and demonstrated a much greater loss in lean body mass and a dramatic decline in muscle insulin sensitivity.

2. We have completed the study in which 7 subjects received an essential amino acid/carbohydrate supplement (EAA group) and 6 subjects received a placebo (Placebo group). The findings indicate that supplementation with EAA maintained lean body mass (LBM) throughout 28 days of bed rest, while a placebo group experienced a loss of LBM. The EAA supplement maintained LBM by stimulating net muscle protein synthesis to a much greater extent than meal ingestion alone. Although this stimulation is diminished with increased inactivity, the net gain in muscle protein is still significantly greater than that produced by meals alone. In other words, even though the anabolic response to the EAA supplement decreases after 28d of bed rest, it is still capable of producing a significant increase in net muscle protein synthesis.

Though EAA supplementation is capable of maintaining LBM, it does not fully maintain muscle strength. While the loss of muscle strength was twice as great in the placebo group, the EAA group also lost leg muscle strength after 28d of bed rest despite the preservation of leg lean mass. These findings demonstrate that the maintenance of LBM alone is insufficient to fully maintain muscle function.

In a concurrent investigation we also demonstrated that ingestion of an amino acid and carbohydrate supplement does not results in a subsequent compensatory nadir in net phenylalanine balance and does not affect the normal anabolic response to ingestion of a nutritionally mixed meal.

Our findings also demonstrate that an EAA supplement is capable of stimulating net protein synthesis during an acute and chronic periods of hypercortisolemia; a model mimicking the stress response to spaceflight. We noted that hypercortisolemia amplifies the loss of muscle protein during the post-absorptive state both before and after 28 days of bedrest.

Though the EAA supplement can slow this loss, it only does so temporarily, such that within 1-2 hours after the supplement, the muscle protein balance is again negative. After 28 days of inactivity, the response to a meal during elevated cortisol is further diminished, such that the muscle is dramatically catabolic. The EAA supplement is not capable of eliciting an anabolic response in the muscle after 28 of bed rest. On the contrary, when the supplement is given without the presence of cortisol, the net effect is muscle anabolism over the study time period.

We have also investigated the effects of 28 days of bedrest and hypercortisolemia, on lean mass and skeletal muscle protein metabolism. Our findings indicate that with elevated cortisol, the loss in muscle mass is almost 3-fold greater than with inactivity alone. In addition, the presence of elevated cortisol induces insulin resistance. After 28 days of inactivity and elevated cortisol, the effectiveness of insulin on skeletal muscle glucose uptake and protein metabolism is greatly diminished.

3. Our findings demonstrate that the anabolic response to a meal diminishes with prolonged inactivity and a stress challenge. The stimulation of net muscle protein synthesis immediately following each EAA supplement translates to a maintenance of muscle protein over a 24 hr period, and in turn, over the 28 days of bed rest. However, the maintenance of LBM does not entirely translate to maintenance of muscle strength. The interaction of inactivity and stress exacerbates the ineffectiveness of ordinary meals. Though the EAA supplement can offset muscle catabolism during the stress state, the response is transient and incapable of ameliorating the overall loss of muscle protein. Taken together, these findings indicate that a nutritional supplement alone can reduce the muscle atrophy associated with space flight. However, whereas muscle mass can be maintained with a specified nutritional intervention, other modalities are required to preserve muscle function.

The catabolic interaction of inactivity and hypercortisolemia is particularly applicable to a patient population where trauma or major surgery results in prolonged inactivity and a stress response. This combination is particularly deleterious to skeletal muscle resulting in a dramatic loss of muscle mass and function, in great part due to the accompanying insulin resistance.

4. We have completed this project and are currently studying the effects of chronically elevated cortisol and hypocaloric diet throughout 14 days of bed rest on muscle protein, LBM, and muscle function. As of this writing, we have completed 10 subjects. Preliminary evidence indicates that combined elevation of cortisol and a hypocaloric diet throughout bed rest increases muscle resistance to the action of insulin and increases the loss of lean body mass. This investigation is relevant to both clinical and astronaut populations, as both are prone to under-nutrition during a stress state. Further, we intend to investigate nutritional and exercise countermeasures with this model to determine an optimal operational countermeasure that can be economically (in terms of crew time and payload) utilized to ameliorate muscle loss during prolonged space flight.