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by: Wen Zhang

Part I: A Primer on Protein Synthesis

Introduction

The goal of any well-designed resistance training and diet program is to maximize skeletal muscle protein synthesis while minimizing fat gain. In an ideal world, one would be able to maintain a consistently high muscle gained:fat gained ratio. This unfortunately is rarely the case due to a number of physiological and environmental factors. As a result, the average trainee finds him or herself at a point where the quantity of skeletal muscle hypertrophy is no longer satisfactory relative to the amount of adipose tissue gained.

To counter the above scenario, concepts such as nutrient partitioning have become widespread among dedicated trainees. To those less familiar with the idea of nutrient partitioning, the idea at its most basic is to manipulate the direction of nutrient flow such that the majority of ingested nutrients feed skeletal muscle growth and recovery while a minimum is made available to adipose tissue. Because net long-term anabolism requires a certain quantity of nutrients above and beyond those required to maintain tissue homeostasis, positive partitioning is clearly an important requisite in attaining desired results.

Many powerful partitioning techniques exist, with modulation of training and diet parameters and ingestion of compounds (both dietary and pharmaceutical) being the most popular. No matter the approach, or combination of approaches chosen, a crucial requirement in partitioning is to strike a balance between anabolic and catabolic processes.

There is little doubt that high-intensity exercise is one of the most potent stimulators of positive partitioning currently known. However, high-intensity activities inflict heavy tolls on the body, necessitating adequate rest and nutrition to be sustainable over the long-term. Research has shown that during, and possibly immediately following intense exercise, the body is in a state of net catabolism in every respect, including protein synthesis [1-3]. Yet repeated bouts of intense exercise do promote skeletal muscle growth so long as said exercise is not excessive to the extreme, and fuel is consumed afterwards in sufficient quantity [4-9].

In response to the catabolic effect during exercise, many have recommended that pre, during, and/or post-exercise meals be consumed to offset said response and maximize the anabolism which follows. These formulations generally consist of a certain ratio of rapidly absorbed carbohydrates and proteins in solution; the working hypothesis behind such drinks is that the carbohydrates will promote an insulin spike in addition to providing substrate for glycogen replenishment. The insulin spike enhances nutrient uptake into insulin-responsive tissues and antagonizes catabolism while ramping up anabolic processes. Indeed, extensive research has demonstrated these formulas can indeed promote net protein synthesis versus not ingesting anything at all, or solely consuming carbohydrates, harnessing the positive partitioning that occurs after high-intensity exercise [6-7, 10].

Having established that intense exercise sets the body up to optimally utilize nutrients, the natural questions which follow concern optimization of the nutrient ratios and quantities in the pre/during/post-exercise meal. Since the original research in the 90’s which showed that a small quantity of carbohydrates + amino acids had a pronounced effect on net protein synthesis post-exercise, many variants have been proposed and tested[6]. While it is of scientific interest to hypothesize and infer supposed benefits of more exotic cocktails, one must question the point at which the benefits outweigh the cost and/or inconvenience of concocting a high-tech everything-but-the-kitchen-sink formula, versus a boring but tried-and-true carbohydrate-protein drink. There is also considerable value in maximizing the anabolic response with a minimum of calories for those prone to fat gain and/or cutting fat.

In the following article, and those which follow, we will examine current research regarding the specifics of skeletal muscle protein synthesis due to resistance and other high-intensity exercise. Hopefully, a combination of studies which look at the effects of intense exercise on protein synthesis at the molecular, cellular, and physiological level during and after exercise will bring some clarity to this issue.

Overview of Protein Synthesis

Protein synthesis for our purposes will focus on the assembly of amino acid polymers (peptides and proteins) from a single-stranded template called messenger ribonucleic acid (mRNA). This process is called translation, a fitting term considering that the “message” as coded by the mRNA must be decrypted by the protein synthetic machinery in order to place amino acids in their proper sequence to ultimately synthesize a functional protein. RNA involved in protein translation comes in three different flavors: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).

  • mRNA: Messenger RNA is the RNA that serves as a template which dictates just exactly which amino acid will come next in the peptide chain. The actual process of adding amino acids to a growing peptide chain is called translation. An easy way to understand this is that RNA is composed of four different sugars: A, U, G, and C. Each amino acid is coded by different combinations of these four sugars. Therefore, the code for leucine is UUA, while CAU specifies histidine.mRNA is simply a string of this code- hence it is often referred to as a template.
  • rRNA: Ribosomal RNA is the actual machinery that reads the mRNA. As stated previously, mRNA is a template that specifies the number and order of amino acids in a peptide chain. By itself, mRNA is incapable of protein synthesis, though. This is where the other forms of RNA come in. rRNA serves as both the physical platform for protein synthesis, as well as the motor that move along the mRNA and “reads” the code.
  • tRNA: Transfer RNA is the means by which amino acids are physically brought into the vicinity of the rRNA and mRNA. In the cytosol, where protein synthesis takes place, amino acids are essentially floating around. There needs to be a means to bring recognize and bring them in proximity to the rRNA so that they can be inserted into the growing peptide chain.

There are three main steps in the actual process of protein synthesis: initiation, elongation, and termination. Initiation involves the initial assembly of the ribosome, mRNA, and an initiator amino acid brought in by tRNA. Elongation begins with the culmination of initiation, and as its name implies, entails the addition of amino acids to the growing peptide chain. When the mRNA has been “read” to its end, synthesis of the nascent peptide ends in the termination step. While all three processes are indispensable in translation, initiation has been shown to be rate-limiting, and therefore will our focal point [10-14].

Translation Initiation

Translation initiation is mediated by approximately twelve (currently) identified eukaryotic initiation factors (eIFs). The order of events:

    1. Formation of a pre-initiation complex. In this step, one half of the ribosome, the 40S subunit, combines with two eIFs, eIF-1 and eIF-3. This step essentially preps one out of the three necessary components for protein synthesis (the ribosome).
    2. Another initiation factor (not bound to the 40S subunit), eIF-2, is activated (through GTP binding).
    3. The activated eIF-2 binds to a special tRNA carrying an activated amino acid that will be the first one in the peptide chain. This step sets up the second necessary component in translation, the transfer RNA (tRNA).
  1. The eIF-2-tRNA complex binds to the 40S subunit of the ribosome as mentioned in step #1; the activated eIF-2 facilitates both the binding of the tRNA and association with the 40S ribosome subunit (mediated by associated eIF-1 and eIF-3). The preinitiation complex now contains two of the three necessary components for initiation- half of the synthetic machinery (the 40S ribosome subunit) and the tRNA with its amino acid have been brought together; all that remains is to bind the mRNA (the code) and bring in the other half of the ribosome (the 60S subunit) to complete initiation.
  2. mRNA is prepared for interaction with the preinitiation complex through the concerted actions of several other initiation factors. Eukaryotic mRNA contains a structure on one end called a “cap” and this is recognized by a key initiation factor, eIF-4E. eIF-4E is normally inactive due to being bound by an inhibitory protein (eIF-4E-binding protein, or eIF-4E-BP for short); upon stimulation, eIF-4E is freed and forms a complex with eIF-4G and eIF-4A; eIF-4G is thought to serve as a physical scaffold for the organization of the three initiation factors, while eIF-4A mediates mRNA binding by unwinding the mRNA in a region of secondary structure near the cap. When these three factors are organized in this manner, they are called the eIF-4F complex.

To summarize this step:

  1. eFI-4E, whose function is recognition/targeting to the cap of mRNA, is freed from inhibition from its binding protein (eIF-4E-BP).
  2. The freed eIF-4E binds two other initiation factors: eIF-4A, which is necessary for mRNA binding due to its ability to unwind the mRNA, and eIF-4G, which is a physical scaffold that mediates the association of the three initiation factors as well as binding to the preinitiation complex.
  3. The formation of the ternary (three-component) complex consisting of eIF-4E, eIF-4G, and eIF-A is called the eIF-4F.
  1. The final step is binding of the remaining ribosome subunit, the 60S subunit, to form an active synthetic machine (the 40S + 60S subunit coming together results in the 80S complex). This step requires the inactivation of eIF-2; eIF2 dissociates and is reactivated by eIF-2B, and can then participate again in promoting initiation.

The Eukaryotic Initiation Factor-4E (eIF-4E)

The rate-limiting step in protein synthesis, as mentioned, is initiation. The rate-limiting component of initiation appears to be eIF-4E. Accordingly, a great deal of research has focused on the regulation of this factor, and many interesting conclusions can be drawn from interpretation of the data. As is the case with most proteins, eIF-4E is regulated at the level of gene transcription, reversible phosphorylation, and association with inhibitory binding proteins.

The importance of eIF-4E has been demonstrated in studies where it has been inactivated, leading to a marked inhibition of growth to the tune of a 4-7x inhibition in a dose-dependent manner, such that cell death eventually results from gross suppression of eIF-4E[15]. Conversely, the overexpression of eIF-4E has been shown to alter cellular growth and proliferation, often leading to a cancerous phenotype [16, 17]. An interesting phenomenon with respect to eIF-4E overexpression is that the expression of a certain subset of proteins is markedly enhanced [16-18]. The model of strong vs. weak mRNAs has been used to interpret this finding. As mentioned previously, all mammalian cells have a structure on one end of their mRNA called the cap; this is the structure that the eIF-4E binds to. Near the cap is a region of secondary structure (folded mRNA); the extent of folding in this region as well as its length has been demonstrated to influence the ease with which the message coded by the mRNA is translated.

Approximately 90% of mRNAs are classified as “strong,” meaning that they can be translated even in the presence of relatively low levels of eIF-4E. The remaining 10% of mRNAs are “weak,” and these are the subclass which reacts most dramatically to augmentation of eIF-4E [17]. Incidentally, the “weak” mRNAs tend to code for proteins involved in cell growth and proliferation; this is significant, as cells must constantly guard against extremes in protein degradation vs. synthesis lest the die or become cancerous, respectively.

Having a mechanism that allows mRNAs which code for proteins which must be upregulated with ease and rapidity (i.e. enzymes involved in metabolism) to do so without stimulating cell growth/proliferation mitigates survival without promoting undue growth. In this manner, proteins which direct cell cycle progression and growth remain quiescent until a stimulus of appropriate intensity and/or duration allows them to be expressed; under normal circumstances, this means that only under ideal conditions will cells expend the energy to grow and/or divide. Going back to the studies where eIF-4E was overexpressed, a cancerous phenotype is consistent with the upregulation of this 10% population of “weak” mRNAs, as a characteristic of cancerous cells is dysregulation of normal checkpoints in cell cycle progression, leading to runaway growth and/or proliferation [17].

Coming Up in the Next Installation

Having established the importance of initiation, and more specifically, the key role of the eIF-4E in controlling initiation, the next logical step is to explore the factors controlling eIF-4E activity. With respect to skeletal muscle hypertrophy, a certain amount of cell growth is necessary, and therefore we would logically expect that hypertrophy will require a certain level of eIF-4E stimulation above and beyond that necessary to simply maintain the intracellular status quo. We will also look at signals that govern the activity of other important components in initiation, but as will be seen, almost all signals that activate initiation factors involve eIF-4E. Finally, we will examine how dietary and exercise interventions affect protein synthesis at the level of initiation, allowing for practical application of our intellectual efforts.

References

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3. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. (2002) AMPK suppresses protein synthesis in rat skeletal muscle through down regulated mTOR signaling. J Biol Chem, 277(27): 23977-90.

4. Anthony JC, Lang CH, Crozier SJ, Anthony TG, MacLean DA, Kimball SR, Jefferson LS. (2002) Contribution of insulin to the translational control of protien synthesis in skeletal muscle by leucine. Am J Physiol Endocrinol Metab, 282:E1092-E1101.

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12. Kimball SR, Jefferson LS, Fadden P, Haystead TA, Lawrence JC Jr. (1996) Insulin and diabetes cause reciprocol changes in the association of eIF4E and PHAS-1 in rat skeletal muscle. Am J Physiol Cell Physiol, 270(39): C705-9.

13. Kimball SR, Horetsky RL, Jefferson LS. (1998) Implication of eIF2B rather than eIF4E in the regulation of global protein synthesis by amino acids in L6 myotubes. J Biol Chem, 273(47): 30945-53.

14. Kubica N, Bolster DR, Farrell PA, Kimball SR, Jefferson LS. (2005) Resistance exercise increases muscle protein synthesis and translation of eIF2Be mRNA in a mTOR dependent manner. J Biol Chem, 280(9): 7570-80.

15. De Benedetti A, Joshi-Barve S, Rinker-Schaeffer C, Rhoads RE. (1991) Expressionon of antisense RNA against eIF4E mRNA in HeLa cells results in lengthened cell division times,diminished translation rates,and reduces levels in both eIF3E and the p220 component of eIF4E. Mol Cell Biol, 11(11): 5435-45.

16. Graff JR, Zimmer SG. (2003) Translational control and metastatic progression-enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs. Clin Exp Metastasis, 20: 265-73.

17. De Benedetti A, Graff JR. (2004) eIF4E expression and its role in malignancies and metastases. Oncogene, 23: 3189-99.

18. Bjornsti MA, Houghton PJ. (2004) Lost in translation- dysregulation of cap-dependent translation and cancer. Cancer Cell, 5: 519-23.

 

The Role of AMPK in Skeletal Muscle Protein Synthesis During Exercise 
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