Abstract

The purpose of this article is to investigate and evaluate the effects of amino acid supplementation in combination with resistance exercise on muscle hypertrophy and strength. The mechanisms of different processes in protein turnover on which amino acid supplementation has an effect are explored. Amino acid supplementation in combination with resistance exercise increases serum IGF-1 concentrations, decreases testosterone concentrations and leaves myostatin expression unchanged. These processes increase mRNA translation, which results in an increase in hypertrophy and, if resistance training intensity is high enough, in increases in strength. In the end it seems that although not all effects of amino acid supplementation are positive for protein synthesis, the net effect is an increase in protein synthesis.

Introduction

Many athletes engaging in sports which require explosive force or great strength, use amino acids as nutritional supplements to increase muscle mass and strength or recover from training more quickly. It is generally known that amino acids are necessary for an increase in muscle mass, functioning as substrates for protein synthesis. In addition it is assumed that athletes need more amino acids than normal sedentary persons and therefore would do well to use amino acid supplements to improve their performance (Lemon, 2000). Many companies producing supplements support this claim, however, the basis for this claim is not so evident. In addition, it is less well known what the effects of amino acid supplementation are on the mechanisms which lead to an increase in muscle strength and fibre size (reference). The purpose of this study is to investigate if there are scientifically proven ergogenic effects of amino acid supplementation on muscle strength. In addition, it will be investigated if amino acids have an effect on mechanisms regulating muscle strength and protein synthesis and degradation, such as mRNA translation. Furthermore, the effects on hormonal factors such as testosterone, insulin-like growth factor 1 (IGF-1) and myostatin will be investigated because these are important regulators in muscle hypertrophy (Harridge, 2007). To understand the effects amino acid supplementation might have, the different mechanisms in protein turnover are first explained. In this study, muscle strength is defined as the greatest amount of force someone can exert at one time. Because there are many different amino acid supplements available, only the effects of whey amino acids will be evaluated to avoid interference from other possible effects of amino acids, derived from different sources (Hofman & Falvo 2004) and because most research regarding amino acids has been performed with whey amino acids.

Amino acids in protein turnover

From amino acid to protein Proteins fulfil many roles but are best known as the building blocks of the cells in the in the human body. In a healthy living cell there is always a balance between protein synthesis and breakdown, or protein turnover. If the rate of protein synthesis is higher than the protein breakdown, the cell will hypertrophy, if the rate of protein synthesis is lower the cell will atrophy.  Protein turnover is regulated by different signals given to the cell, which will be described later.

 

 

 

 

 

 

 

 

 

 

Figure 1: Overview of the processes involved in protein synthesis (from Jaspers, 2007)

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2:Translation of a mRNA molecule (from: Alberts et al. 1998).

If a cell receives the signal to increase protein synthesis, specific genes in the DNA coding for proteins that induce hypertrophy, are copied to a sequence of mRNA by the enzyme RNA polymerase in a process called transcription (Alberts et al. 1998) (for an overview see Figure 1). These strands of mRNA contain the instructions to form protein. After transcription mRNA is transported from the nucleus to the cytosol. Inside the cytosol, the mRNA strand binds with a 40S ribosomal subunit which then localizes the AUG start codon. The 40S ribosomal subunit subsequently releases initiation factors to bind with a 60S ribosomal subunit, forming an 80S ribosomal complex, initiating the elongation phase of protein synthesis (Kimball & Jefferson 2006). The ribosomal complex contains four sites for RNA to bind. One of these sites is for the mRNA molecule and the three others are for tRNA molecules which transport amino acids to the ribosome. The ribosome will connect the amino acids from the tRNA molecules which complement the code on the mRNA to each other. This process is called elongation (see figure 2) and is repeated until the ribosome encounters a stop codon in the mRNA code, which indicates that the protein is finished and subsequently the protein is released in the cytosol.

 

 

 

 

 

 

 

 

 

Figure 3: Ubiquination of a protein (from: Hoeler et al. 2006).

 

Protein degradation Proteasomes are mostly responsible for the breakdown of proteins within the cytosol, a process called proteolysis. Proteins that are damaged or need to be broken down are recognized by specialized enzymes, called ligases. Ubiquitin is first activated by the ubiquitin activating enzyme E1. The activated ubiquitin is then transported to the E2 ligase from which the E3 ligase transports it to the protein that is designated by a lysine molecule and attaches the molecule to the protein (Zhang et al. 2007). The proteasomes then recognize and break these ubiquinated proteins down to amino acids (see also figure 3) by hydrolizing the peptide bonds between the amino acids. There can be a number of reasons why proteins must be degraded. Many proteins are meant to have a short lifespan and have to be degraded quickly but it is also possible that proteins are misfolded during synthesis and have to be degraded as well. Proteins that are misfolded or denatured are also recognized as targets for adding ubiquitin, presumably by uncovered amino acid sequences giving off the signal for adding ubiquitin (Alberts et al. 1998).

 

 

 

 

 

 

 

 

 

 

 

Figure 4: IGF-1 regulation in protein turnover (from: Glass 2005).

IGF-1

Activation of transcription can occur through an increase in serum testosterone or serum IGF-1 (see figure 4) (Urban et al. 1995, Glass 2005). IGF-1 induces increases in strength by binding to Insulin Receptor Substrate 1 (IRS-1), which activates phosphatidylinositol-3 kinase (PI3K)(Urban et al. 1995). PI3K in turn stimulates two different pathways leading to protein synthesis, the AKT/mammalian target of rapamycin (mTOR) pathway, and the AKT/glycogen synthase kinase 3 beta (GSK3-β) pathway (Glass 2005). Activating the AKT/mTOR pathway stimulates p70S6 kinase (p70S6k), which increases the rate of translation and thus increases protein synthesis and simultaneously inhibits PHAS-1, an inhibitor of protein initiation factor eIF4-E. Activating AKT also phosphoralizes GSK3-β, which functions as an inhibitor of the eukaryotic initiation factor 2-B (eIF2-B), also a factor leading to protein synthesis (Glass 2005). In other words, AKT removes one of the brakes on protein synthesis.

Testosterone

The androgenic hormone testosterone is well known for its anabolic effects on muscle size and strength (Herbst & Bhasin 2004, Kadi 2008, Sinha Hikim et al. 2002). Sinha Hikim et al. (2002) found that testosterone induces an increase in the cross-sectional area of the muscle and in the number of myonuclei. Because nuclei in muscle cells can not divide themselves the myonuclei have to come from a different source, indicating that hypertrophy also occurs through increasing the number of satellite cells which then fuse with the muscle fibre. This indication is confirmed by a follow up study of Sinha Hikim et al. (2003). In the study of Sinha Hikim et al. (2004) it is also found that testosterone induces hypertrophy by stimulating the myonuclei to increase androgen receptor expression. This enhances the testosterone binding capacity of the muscle cell and hence inducing hypertrophy (Sinha Hikim et al. 2004).  Testosterone is produced by the Leydig cells in the testes and influences the muscles by binding to the androgenic receptor (AR) at the plasma membrane of muscle cells and satellite cells. When testosterone activates the satellite cells, they show proliferation and since part of those new satellite cells return to quiescence, the total number of satellite cells increases (Kadi, 2008). Satellite cells that do not return to quiescence, can  enter the muscle cell in a process called myonuclear accretion, increasing the number of nuclei which can produce mRNA in the muscle. The advantage of more nuclei is that the muscle fibre is no longer limited to the mRNA production capacity or service area of a smaller number of nuclei, increasing the capacity for hypertrophy. Testosterone also stimulates the myonucleus to increase the rate of transcription by binding to the AR. The AR  then translocates to the hormone-responsive element in the nucleus and binds to the genes responsible for an increase in transcription (Kadi, 2008).

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5: Mechanisms of testosterone action on skeletal muscle (from: Kadi 2008).

Testosterone has different effects leading to muscle hypertrophy. It can activate the myonucleus, stimulating mRNA transcription, or it can stimulate satellite cells to fuse with an existing muscle fibre which causes its capacity for hypertrophy to increase (Kadi 2008).

Another effect of testosterone in humans is that it improves the efficiency of protein reutilization. In a fasted state, protein balance is usually negative and the amino acids derived from breakdown are transported outside the cell into the bloodstream. Testosterone however inhibits the transport outside the cell and the muscle reutilizes the amino acids to form proteins (Ferrando et al. 1998).

Myostatin

One of the factors controlling muscle protein breakdown is myostatin. Myostatin negatively regulates stem cell activation (Bradley et al. 2008) by binding to receptors that are responsible for activating proliferation in satellite cells, limiting accretion of myonuclei and by doing so, limiting the capacity of the muscle fibre to hypertrophy. In addition, it binds to the ActRIIb receptor inhibiting protein synthesis through PI3K/Akt/GSK3-β pathway. (Yang et al. 2007). In essence it is an antagonist to IGF-1, instead of activating the PI3K/AKT/GSK3/ GSK3-β pathway, it inhibits it (Ji et al. 2008, Yang et al. 2007). Myostatin functions by upregulating the P21 CDK inhibitors which causes the cell cycle of the myoblasts to stop in G1, resulting in less myoblasts available for differentiation in myotubes necessary for hypertrophy (Sharma et al. 2001).

 

 

 

 

 

Myf5 and MyoD designate precursor cells to develop into myoblasts after which they proliferate and differtiate.

A: Functional myostatin increases p21 activity which inhibits cyclin-E.Cdk2. This decreases Rb activity and stops the cell cycle in G1, resulting in less committed myoblasts and therefore less myotubes.

 

 

 

 

 

 

B: Without functional myostatin, Rb activity does not decrease and the cell cycle of the myoblast is not stopped which results in a larger number of myotubes.

Figure 6: schematic explanation of myostatin (from: Thomas et al. 2000).

 

 

 

 

 

 

 

 

 

 

 

 

Figure 7: Myostatin as an antagonist of IGF-1 (from: Yang et al. 2007).

Myostatin inhibits two actors in the PI3K/AKT/GSK-3β pathway, decreasing protein synthesis.

Effects of Amino Acid supplementation on mRNA translation

In addition to having a wide range of effects on muscle protein synthesis mediators such as IGF-1, Testosterone Myostatin and their function as substrates for protein synthesis, amino acids can also function as a signalling substance, stimulating protein synthesis. According to Kimball & Jefferson (2006), amino acids activate, among others, mTOR which increases mRNA translation. This is supported by Bolster et al. (2004), who concluded that amino acids increase mRNA translation by directly stimulating eIF2B and acting on different components in the PI3K/AKT pathway rather than by activating it completely. The mechanism by which amino acids act upon these components remains largely unknown

Vary et al. (1999) studied the effect of supraphysiological doses of amino acids on translation initiation in rats. They also found that high availability of amino acids resulted in an increase in mRNA translation through increased binding activity of eIF4E by increasing the amount of active eIF4E•eIF4G complex. In contrast with the other studies, eIF2B activity remained unchanged. Although these studies all indicate that amino acids stimulate the rate of mRNA translation and thus increase protein synthesis, this is all tested in laboratory studies. To date, no studies have been performed on the effect of amino acid supplementation on mRNA translation in healthy athletes. However, in the study of Fujita et al. (2007) the effect of a leucine-enriched essential amino acid–carbohydrate mixture on active young healthy subjects was investigated. It was found that mTOR signalling to P70S6K1 and 4E-BP1 was stimulated by the supplement. It was concluded that this effect was due to increased availability of amino acids. Although this study was not performed with a pure amino acid supplement, the result is in line with those reported by others (Vary et al. (1999), Bolster et al. (2004) and Kimball & Jefferson (2007)). Also, Kadowaki & Kanazawa (2003) described pathways through which not only mTOR is stimulated directly by the amino acid leucine, but in which leucine inhibited protein degradation as well (see figure 8). However, details about these pathways still remain unknown. In conclusion, amino acids stimulate mRNA translation by activating mTOR and possibly by inhibiting protein degradation.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 8: Insulin and amino acid (leucine) pathways in protein turnover (from: Kadowaki & Kanazawa 2003). Leucine has an inhibiting effect on processes that increase protein degradation, such as the activity of autophagosomes which degrade cell components. In addition leucine directly stimulates mTOR increasing protein synthesis.

Effects of amino acid supplementation on hormones in protein synthesis and degradation In this chapter the effects of amino acid supplementation on the concentrations of the aforementioned hormones are reviewed.

IGF-1

According to a study under undernourished infants from Pucilowska et al. (1993) IGF-1 plasma serum levels increased from very low levels of 24 and 32 ng/ml, to 160 and 322 ng/ml, in a normal protein and high protein intake group respectively. Altough this study was performed on malnourished young children, it indicates that protein intake influences IGF-1 concentrations and that a higher protein intake results in higher IGF-1 concentrations in the blood plasma. In a study by Willoughby et al. 2007, 19 untrained healthy males were divided in two training groups, one group receiving a whey amino acid supplement, the other receiving a carbohydrate placebo. After completing the strength training protocol, IGF-1 serum levels of both groups were significantly higher than at baseline. However, the group that received amino acid supplementation had an IGF-1 serum concentration that was more than twice as high than the concentration in the placebo group, 364.00±109.20 pg/ml versus 153.00±45.90 pg/ml respectively. This is in contrast to a study of Levenhagen et al. (2002), who did not report any differences in IGF-1 serum concentrations after supplementation with a combination of carbohydrate and whey protein. The subjects participating in this study were tested three times receiving a supplement directly after finishing the test exercise, but did not perform any supervised strength training. They were only encouraged to continue their normal daily living pattern. This study indicates that serum IGF-1 levels will not increase through amino acid supplementation alone. In light of the subjects only performing a 60 minute cycling exercise at 60% VO2 max, but no high intensity strength training, such as in the study of Willoughby et al. 2007, who did find an increase in serum IGF-1 in subjects with amino acid supplementation, it may be necessary to combine amino acid supplementation with strength training to maximally increase serum IGF-1 levels. From the studies above it can be concluded that amino acid supplementation increases plasma serum IGF-1 concentrations. However, it must be kept in mind that it remains unknown what the effect of amino acid supplementation is on intracellular IGF-1 levels.

Testosterone

Hulmi et al. (2005), investigated the effect of whey protein taken 30 minutes before a strength training session on testosterone concentration and compared these results with a placebo. All subjects were tested while receiving the whey protein supplement as well as the placebo, but were unaware which they received. The subjects were all healthy young adult strength trained male athletes. It appeared that after the exercise protocol, serum testosterone concentrations were significantly lower when amino acid supplementation was used compared to the placebo. In a study by Chandler et al. (1994), experienced male weight lifters were given a whey protein immediately and two hours after the strength training protocol. They found a decrease in serum testosterone concentration after completion of the training, both after the water placebo and the whey amino acid supplement, but the decrease after amino acid supplementation was greater. Chandler et al. (1994) hypothesized that if the lower serum testosterone concentration were due to a lower production, luteinizing hormone concentrations would also be decreased, but this was not found in their study. Further possibilities such as decreased sensitivity of the testes to luteinizing hormone or increased testosterone uptake by the muscles were unfortunately not investigated in this study. In contrast, Kraemer et al. (2006) found no decreases in serum Testosterone concentrations in the group that received amino acid supplementation. After two weeks however, serum testosterone concentrations of the group receiving amino acids differed significantly with the testosterone concentrations of the placebo group, which was decreasing during the training period. This would indicate that amino acid supplementation is beneficial for the serum testosterone concentration. The difference in results with the previously described studies can lie in differences in protocol between the studies. The training protocol in the studies of Hulmi et al. (2005) and Chandler et al. (1994), allowed the subjects to recover from training. The training protocol in the study of Kraemer et al. (2006) involved heavy resistance training on consecutive days during four weeks. Interestingly, in an earlier study of Kraemer et al. (1998) subjects performed heavy strength training for three consecutive days and it was found here as well that when the subjects were provided with amino acids, serum testosterone concentrations were significantly lower compared with a placebo. Although they used a different amino acid supplement in this study than in the previously described studies, the results are the same as in the studies of Hulmi et al. (2005) and Chandler et al. (1994). Although there is still controversy over the effects of amino acid supplementation, it would seem that amino acid supplementation lowers the serum testosterone concentration unless very heavy strength training is performed. However, the decrease in serum testosterone does not need to be detrimental to the anabolic effects of training, because this effect can also be caused by increased uptake of testosterone by the muscles. Amino acids might facilitate testosterone uptake by the muscles, which increases protein synthesis, but this also results in a decrease in serum testosterone concentration (Chandler et al. 1998).

Myostatin

To date, only one study has been performed on the effects of whey amino acids on myostatin expression in humans. Hulmi et al. (2008) provided resistance trained older men with either a whey protein supplement or placebo before and after the resistance training for a period of five months. The results from this study indicated that, contrary to what might be expected, amino acid supplementation prevents the decrease in myostatin expression, as demonstrated by Hulmi et al. (2007), usually found after heavy strength training. Hulmi et al. (2008) explain this by stating that the decrease in myostatin after strength training without amino acid supplementation is caused by a mechanism which prevents further muscle damage. Although this effect does not seem beneficial, the decrease in myostatin expression might not be needed when there are sufficient amino acids available to increase protein synthesis after training, which results in an unchanged myostatin expression.

Effects of Amino Acid supplementation on muscle strength and muscle size

Andersen et al. (2005) reported 18%± 5% and 26%±5% increases in cross sectional area in the Type I and Type II fibers of the vastus lateralis muscle in the group that received protein, while no changes occurred in the group that received a carbohydrate supplement. In addition, vertical squat jump height increased 9%±2% in the protein group versus no change in the carbohydrate group and countermovement jump height did not differ significantly between groups, 10%±2% versus 7%±6%. Surprisingly there where no differences between groups in voluntary peak torque, which means that the group that received protein was not able to generate much advantage from their increased muscle size. It is proposed that the lack of difference is caused by neurological adaptations in the carbohydrate group, which would seem enough to counter the advantage of the larger muscles in the protein group. An other reason could be that the subjects were tested differently than they were trained, but in the study from Hoffman et al. (2006) where subjects were tested with the same exercises, no relationship was found between the amount of protein in a diet and strength increase. Kraemer et al. (2006) tested subjects with resistance training experience but, after a four week training programme, did not find any differences in strength between the amino acid or placebo receiving group. However, the placebo group displayed a significant loss of strength after one week of training, which was not seen in the amino acid group. The placebo group experienced strength decreases of -5,1±1kg in 1RM squat weight and -3,4±0.5kg in 1RM bench press weight. This result implies that amino acid supplementation counters the decrease in strength in the recovery phase after intensive training, and therefore that it can help increasing training intensity and strength. However, training intensity was apparently not kept high enough during the course of this study, because then it is to be expected that the strength of the placebo group would keep decreasing, whereas the amino acid group would still demonstrate strength increases. This is confirmed by a study by Willoughby et al. (2007) in which subjects had to perform a long term high intensity training programme and received a carbohydrate or amino acid supplement. After completing the training, all subjects had increased their strength and muscle mass. However, the group receiving an amino acid supplement increased their 1RM bench press by 48%±2% of the subjects’ body weight and their 1RM leg press by 113%±12% of the subjects’ body weight versus 20%±8% and 61%±3% in the carbohydrate group respectively. From the results of the studies above it can be seen that amino acid supplementation stimulates hypertrophy and, if the strength training intensity is high enough, increases strength further than the same regime without amino acid supplementation.

Discussion

Although there is still much debate over the effectiveness of amino acid supplementation in increasing muscle mass and strength, this is probably mainly due to differences in testing methods. From the studies described above, it can be seen that amino acid supplementation in combination with strength training, increases blood plasma IGF-1 concentrations (Willoughby et al. 2007) and increases the amount of mRNA translation (Bolster et al. (2004), Gingras et al. (2001)), which can be a direct effect of the increased IGF-1 plasma concentration (Glass, 2003). Amino acid supplementation also has an apparent negative effect on protein synthesis. Serum testosterone concentration decreases more after resistance training when amino acid supplementation is used than when a placebo is used (Hulmi et al. 2006, Chandler et al. 1994). There can be different reasons why serum testosterone concentration decreases such as, as already pointed out by Chandler et al. (1994), increased uptake by the muscles or decreased luteinizing hormone sensitivity. An other possibility is that amino acid supplementation allows the subjects to keep up an higher intensity training programme which results in a greater decrease of testosterone afterwards, although this was not tested in any of the longer term studies.  An other surprising finding was that myostatin expression was not down regulated after strength training when amino acid supplementation was used, whereas it did decrease in the placebo group (Hulmi et al. 2008). This was explained by the fact that down regulation of myostatin is a safety measure for when muscles are damaged, to prevent any further protein degradation. This would not be necessary when amino acids are sufficiently present. However this effect could also be caused by the age of the subjects, because the study was performed on older men. Perhaps the effects of amino acids on myostatin expression in young adult resistance trained men are different.  In addition, it was seen that the amino acid leucine directly stimulates pathways leading to hypertrophy and it inhibits processes that play a role in atrophy (Kadowaki & Kanazawa 2003). However, the effects of leucine or other amino acids that might have a direct effect on pathways leading to muscle hypertrophy or strength, have not yet been investigated. It would be interesting to study the effects on cultured muscle fibres such as in Jaspers et al. (2008), because other interfering factors can be ruled out and the mechanisms involved can be studied effectively. Finally, it can be seen that the effects of amino acid supplementation described above, and undoubtedly many which remain undescribed, result in an increase in hypertrophy and, although this is not found in every study (Andersen et al. 2005, Hofman et al. 2006), in an increase in muscle strength (Willoughby et al. 2007). It is likely that amino acid supplementation is only useful when the intensity of the training is high enough and when the regular diet pattern does not suffice anymore, which is uncommon for almost all in western society but the most elite athletes (Lemon, 2000).  Other than of what use amino acid supplementation might be, it is important to know what the risks of amino acid supplementation are. Lawrence & Kirby (2002) state that excess protein is converted into urea and excreted and therefore athletes consuming excessive quantities of protein, run the risk of becoming dehydrated, calcium loss, liver and kidney damage, diarrhea and contracting gout. However, according to Tipton & Wolfe (2004) there is no evidence for kidney damage in healthy athletes with no underlying renal disease. It was stated by Tipton & Wolfe (2004) that excessive protein ingestion can be consumed at the expense of other necessary nutrients, which could be detrimental for both health and performance. Moreover, there may be risks associated with the effects of amino acid supplementation on IGF-1 or testosterone levels. High IGF-1 levels have been demonstrated to be an important risk factor for developing prostate cancer (Wolk et al. 1998). However, outside these potential health risks and beneficial effects in muscle anabolism, whey amino acid supplementation also has beneficial health effects, such as improving the immune function and gastrointestinal health (Ha & Zemel 2003).  Protein turnover is, as can be seen from the mechanisms depicted above, a complicated system with many interactions. Not only are amino acids substrates for protein synthesis, but some amino acids also have regulatory functions (Kimball & Jefferson 2006). In addition, amino acids also influence hormonal factors such as IGF-1, testosterone and myostatin which also influence each other (Urban et al. 1995 Yang et al. 2007). However the extent of these and other interactions is still largely unknown.  In the end, it can be concluded that amino acid supplementation is particularly useful for people who want to increase their muscle mass such as body builders, because it stimulates muscle fibre hypertrophy. In addition, it is also useful for athletes who need great strength in their respective sports, because with a high enough training intensity amino acids have been shown to help improving strength even further.

Acknowledgements

I would like to thank Richard Jaspers for his contribution to this article. His expertise and enthusiastic guidance aided greatly during the writing of this review.

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