A comprehensive approach to describing protein turnover in lambs

60 INTRODUCTION 62 MATERIALS AND METHODS 64 iii RESULTS 74 DISCUSSION 88 ACKNOWLEDGEMENTS 94 LITERATURE CITED 95 GENERAL DISCUSSION 98 LITERATURE CITED 105 ACKNOWLEDGEMENTS 110 1 GENERAL INTRODUCTION Understanding Muscle Growth Through Basic Research Since muscle is essentialiy the end product in meat animal agriculture, one target of basic research has been to understand the nature and control of Its growth. For basic research to yield meaningful applied knowledge, the mechanisms for muscle growth must be understood at the whole-animal, tissue, cellular, subcellular, and genetic levels. Thus, the experimental approach to understanding muscle growth is multifaceted. Several key issues are important: 1) understanding the control and regulation of the growth process; 2) developing appropriate methodologies to measure observed changes that occur in muscle growth and in the metabolic pathways that drive growth; and 3) understanding and interpreting observed measurements in context of the experimental setting and conditions. In the area of control and regulation of growth, growth stimulants have been one of the tools used to understand the metabolic process. The underlying idea in this approach is that by enhancing muscle growth through an exogenous agent, the underlying mechanism of normal muscle growth and metabolism might be more clearly understood. For example, naturally produced hormones in the body such as estrogen, testosterone, and growth hormone have been applied exogenously to animals to produce a growth effect. This suggests that these hormones may play an important role in regulating normal muscle growth. Future application of these regulators in a controlled setting along with measurement of the corresponding changes in metabolic pathways may be an effective means of describing their role In regulating normal muscle growth. Similarly, an exogenous growth stimulant (pharmaceutical rather than natural) might be applied to produce an obvious increase in muscle growth. By measuring the effects of this agent on normal metabolic 2 processes, key regulatory points might be identified and quantitated. Such control points may include natural hormone levels, nutrient energy availability, amino acid supply and composition, and age related changes in gene expression. Thus, metabolic regulators may be very useful as tools in describing normal muscle growth and the processes which contribute to the accumulation of muscle protein. A second key area to understanding the muscular growth process is the development of methodologies to quantify changes in the underlying metabolic pathways. Two of the fundamental pathways which affect muscle growth are protein synthesis and protein breakdown. The underlying assumption is that muscle growth or atrophy results from changing the relative rates of these two opposing metabolic processes. When protein synthesis exceeds protein breakdown, muscles accumulate protein and gain mass. When the rate of protein breakdown exceeds the rate of synthesis, muscle atrophy occurs. Since these processes are an integral part of muscle growth and protein accretion, development of accurate methodologies to measure the rates of protein synthesis and protein breakdown in individual tissues will be fundamental to understanding the muscle growth process and its regulation. Several techniques have been used to study growth at the whole-animal and tissue levels such as nitrogen balance, plasma amino acid flux, and tissue protein synthesis. One problem with these methodologies is that they are often used independently and rarely conskJered in combination. As a result, interpretation of results has been ambiguous. For example, in applying P -agonists to animals, one technique such as plasma amino acW flux may suggest that growth was enhanced by an increase in protein synthesis (indirectly) while another technique such as radioisotope incorporation into mixed muscle proteins may suggest that a decrease in protein breakdown was responsible because protein synthesis was the same among treated animal and 3 controls. Thus, it is difficult to determine which metabolic process, protein synthesis or protein breakdown, was primarily affected by P -agonists and thereby influenced growth. Since much of the previous work in this area has used only a single technique, it is difficult to evaluate whether differing results arise from differences between studies or differences between methods. Thus, it may be useful to compare these techniques under Identical conditions to assess the appropriate use of each technique In describing muscle tissue growth. Knowing the strengths and limitations of each method could, in turn, provide direction for further research in choosing the appropriate method under given conditions as weil as suggesting possible interpretation of ambiguous results. Another methodological consideration in the study of muscle growth Is the development of new techniques to measure the rate of proteolysis in vivo. To date, a valid technique to measure protein breakdown has not been reported. Most of the literature that refers to changes in protein breakdown being responsible for an increase in growth have only Indirectly estimated the rate for breakdown. Protein synthesis can be measured with isotopic tracers and growth rate can be estimated by change in live weight. Thus, the rate of protein breakdown Is estimated indirectly as the difference between synthesis and growth. Hence, it appears that new technology applied to accurately measuring the rate of protein breakdown in muscle would greatly impact the study of muscle growth and its regulation. Given that the appropriate growth regulators and methodologies are available, the importance of experimental context must also be considered. This is especially important in making the transition between isolated muscle cell culture systems and muscle in the context of the whole animal. Tissue culture methods have been developed in order to study specific events In Indivklual tissues without the influence of other organs and metabolites. This approach has the advantage of reducing the apparent complexity of whole-animal events resulting from interaction between 4 multiple organs and cell types to single events in a specific cell type. In addition, these tissue culture studies are relatively Inexpensive relative to whole animal experiments. Thus, muscle tissue culture has been a valuable tool in studying the processes and regulation of muscle growth. The context of these techniques is crucial, however, because while such experiments reveal a great deal about the specific events in muscle tissue, and serve as a great screening tool for potential modulators of protein turnover, the data do not necessarily guarantee the relevance to the whole animal. For example, an agent might be developed to decrease protein breakdown by 50% in muscle cell culture, but have no effects in the whole animal system. Similar arguments can be made relative to measurement of mRNA levels, enzyme activity and concentration, gene expression, and developmental changes in endocrine profiles. The measurement of such parameters may be important in characterizing an isolated system, but do not necessarily indicate if this system is important in the whole-animal setting. Thus, in light of the complexity of the whole animal system, it appears that context is key to determining which events and processes have large influences on muscle growth, and which events have only minor importance. The Wea of experimental context is also Illustrated in terms of nutritional status. Some exogenously applied growth stimulants may only be effective if the animal is compromised nutritionally or is In a state of stress, injury, or disease. Other regulators, however, may only be effective if nutrients are in excess and conditions are optimal for muscle grov/th. On the whole, then, the experimental approach toward understanding the nature and control of muscle growth depends greatly on the ability to modify muscle growth in a predictive fashion with exogenous growth enhancers, the availability of appropriate methodology to measure observed changes In the metabolic changes which support growth, and the interpretation of results in terms of the experimental conditions employed. 5 Goals of the Present Study The present investigation compared many of the current technologies used to describe muscle growth and metabolism. Selected growth enhancers were used as tools to modify muscle growth in a predicted fashion. The resulting changes in muscle growth were then assessed by a variety of methods and in a variety of tissues in order to characterize the overall Importance of protein synthesis and protein breakdown in regulating muscle growth. The specific goals were to: 1. Describe the mechanism of action of p -agonists in stimulating muscle growth. 2. Evaluate the appropriate use of currently used tracer techniques to describe changes in muscle protein accretion. The idea behind the first goal was to use specific exogenous agents to modify growth in order to determine what factors may control muscle growth in normal conditions. Conclusions drawn from this whole-animal and tissue level study should enable scientists to ask appropriate meaningful questions about regulation at the cellular and subcellular levels. For example, If p agonists primarily act by increasing protein synthesis in tissues, it may be appropriate to invest resources to probe specific tissues for specific proteins which are synthesized at a faster rate (myofibrillar proteins, sarcoplasmic proteins, etc.). On the other hand, if a decrease in protein breakdown is the main effect, probing for specific proteolytic enzyme levels and activity may be meaningful. It may also be possible that the main effect of P -agonist on muscle growth is mediated through another organ such as liver. To speculate, the endocrine pathway of p -agonists may target the liver through sp