Research Update #4

by Hypertrophy

As we approach the new millennium we find the science of building muscle progressing faster than ever before. Long gone are the days of simple trial and error when it comes to building muscle. The modern bodybuilder demands more than just "hear say" if they are to adopt a new training routine or nutritional supplement. This column was created to keep today’s bodybuilder on the cutting edge of scientific research that might benefit them in their quest for body perfection.

A quick note before we go on:

I thought a brief discussion of the mechanism by which proteins are synthesized would be in order to better understand the research presented in this month's Research Update. So without further adieu, proteins are made according to the information contained on our genes. A "gene" is a series of nucleotide bases in a strand of DNA. Although the majority of genes contain information for building proteins, some do not, and serve other functions in protein synthesis. The nucleotide bases are arranged in pairs, which, can be read (from mRNA) in sets of three called "codons". Each codon corresponds to a given amino acid. Sequences of codons code for sequences of amino acids which form polypeptide chains. All proteins in the body are composed of one or more polypeptide chains.

Some genes contain information for constructing RNA molecules, which are required for proteins synthesis. There are three kinds of RNA that are needed for protein synthesis, Messenger RNA (mRNA), Transfer RNA (tRNA), and Ribosomal RNA (rRNA). Messenger RNA carries the instructions for building protein from DNA within the nucleus, to the cytoplasm outside the nucleus. Transfer RNA is a translator of sorts that converts the information on mRNA into a corresponding sequence of amino acids. Ribosomal RNA is actually part of the ribosome. It is the site on the ribosome where amino acids are assembled into polypeptide chains.

There are two steps leading from the DNA molecule to polypeptide chains. In the first step, a DNA template is used to create RNA. This process is called transcription. Transcription is accomplished through a series of steps. First, a double-stranded DNA molecule is unwound at a particular gene region, then an RNA molecule is assembled in mirror fashion based on the exposed bases of one of the DNA strands which serves as a template. In eukaryotic cells, new RNA molecules undergo modification into a final form before being shipped from the nucleus out to the cytoplasm. Typically, enzymes attach a "cap" at the 5' end and a poly-A tail at the 3' end. Then non-coding portions (introns) of the RNA are removed and the rest (exons) are spliced back together. The mRNA is not exported until all of this is complete.

The second step is called translation. In this process mRNA interacts with tRNAs and ribosomes in such a way that amino acids become linked one after another. The sequence in which the amino acids are linked together determines what protein will be formed. Translation proceeds through three steps, initiation, elongation, and termination. In "initiation" a portion of the ribosomal complex first binds with an "initiator tRNA" and then with the mRNA molecule. A second portion of the ribosome complex then binds with the first to form the initiation complex. During "elongation", the amino acid chain then begins to elongate as tRNAs deliver more amino acids to the ribosomal complex. The base pairs on the amino acid bound-tRNA match up the base pairs on the mRNA to form specific polypeptide chains. Finally, the chain is terminated once the ribosome reaches the "stop codon" on the mRNA.

References:

Suzuki DT, Griffiths AJ, Miller JH, Lewontin RC (eds): An introduction to Genetic Analysis. 3^rd ed. New York, W.H. Freeeman and Company, 1986.

Starr C, Taggart R (eds): Biology: The unity and Diversity of Life. 6^th ed. Belmont,Ca., Wadsworth Publishing, 1992

Starr C, Taggart R (eds): Biology: The unity and Diversity of Life. 7^th ed. Belmont,Ca., Wadsworth Publishing, 1995

Title: Discovery of nonsteroidal androgens.

Researchers: Dalton JT, Mukherjee A, Zhu Z, Kirkovsky L, Miller DD
Department of Pharmaceutical Sciences, College of Pharmacy, University of  Tennessee, Memphis

Source: Biochem Biophys Res Commun. 244(1):1-4, 1998

Summary: An in vitro study was performed to assess the ability of non-steroidal compounds to bind with the androgen receptor (AR) and activate transcription. The compounds to be tested were derived from the structure of the anti-androgen R-Bicalutamide. The androgen receptors were prepared using the prostate glands of male Sprague-Dawley rats. Transcriptional activation was measured using transfected CV-1 cells.

In order to assess the binding affinity of these compounds, they were incubated in a medium containing androgen receptors. Each compound was tested in increasing concentrations until the minimum concentration that resulted in maximum receptor saturation was attained. Then the experiment was repeated using the high affinity ligand, ³H-MIB. The concentration of each compound needed to completely saturate the androgen receptors was then compared to the amount of ³H-MIB needed to do the same.

The efficacy (i.e. maximal degree of AR-mediated transcriptional activity observed) and potency (i.e. the lowest concentration of ligand able to induce maximal transcriptional activity) were determined by comparing the transcriptional activity induced by various concentrations of each compound to that of dihydrotestosterone (DHT). The efficacy of individual compounds compared to DHT was calculated by dividing the maximal transcriptional activation observed for each compound by the maximal transcriptional activation observed for DHT. All experiments were performed in triplicate. Potency was reported as the lowest concentration of the ligand used during transfection experiments capable of producing maximal androgen receptor-mediated transcriptional activity.

Discussion:

First a few words about the study design. The methods used in this study are common when testing new drugs that bind with receptors. The easiest thing to do is simply put the drug in a medium containing the receptors and see how well they bind to them. Does this mimic an in vivo environment? Not exactly. But it does answer the question as to whether the drug will or will not bind to the desired receptor. In this study, the ability of these compounds to bind to androgen receptors, or their binding affinity, was compared to that of ³H-MIB which is known to have a very high affinity for the androgen receptor. The efficacy, or ability of the compound to stimulate maximum transcriptional activity in intact cells, was compared to that of DHT which is thought to be the dominant compound by which androgenic activity is stimulated in the body. The potency, or the lowest concentration of ligand able to induce maximal transcriptional activity in intact cells, was also compared to that of DHT.

This study is the first to discover compounds that do not share the steroidal structure that are able to bind with the androgen receptor as an agonist for transcriptional activity. Remember that transcription is the first step in protein synthesis. It is sort of ironic that these compounds were created using the structure of known anti-androgens. One particular compound named R-1 had very similar androgenic activity to that of DHT. Considering that testosterone has a binding affinity (to androgen receptors) that is only ~10- 50% that of DHT, where as several of these new non-steroidal compounds had affinities that were nearly identical to DHT, shows that these new compounds have tremendous potential in bodybuilding. Of course, data on dissociation rates were not provided. It is believed that the different dissociation rates of testosterone and DHT are responsible for the greater potency of DHT.

So why create drugs that act like steroids but aren’t steroids? Well, simply put, side effects and control. Many of the unwanted side effects of testosterone and its derivatives are a result of its structure. The ultimate goal in creating a ligand for the androgen receptor for myotropic activity would be to create a drug that has high binding affinity (i.e. similar if not greater than DHT) as well as a slow dissociation rate, and a low rate of hepatic clearance from the system. The inability of these drugs to be converted into estrogen is also a great advantage of their non-steroidal structure. Another advantage is half-life. In an in vitro study you cannot predict half life. However, there is no reason to doubt that the half-life of these compounds could be longer than that of testosterone and its derivatives. This would allow less frequent dosing and perhaps lower dosages. Finally, one must concede that any of the side effects that are a result of simple androgen activity may not be avoidable even with non-steroidal androgens. These side effects would have to be dealt with further down the line, after receptor activation.

Fortunately, the authors reported that they would continue to investigate these compounds as well as other newly synthesized compounds in their laboratory. They even went on to say that there are indications that efficacy and potency could be further increased with slight modifications to their existing structures. I will go out on a limb and say that the first non-steroidal androgenic/anabolic drugs will undoubtedly be a result of this study and those that follow from this lab. Stay tuned to Mesomorphosis for further developments in the developments of these novel anabolics.

Title: Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise.

Researchers: Gautsch TA, Anthony JC, Kimball SR, Paul GL, Layman DK, Jefferson LS
Division of Nutritional Sciences, University of Illinois, Urbana 61801, USA.

Source: Am J Physiol (1998) 274(2 Pt 1):C406-14

Summary: The authors examined the association of the mRNA cap binding protein eIF4E with the translational inhibitor 4E-BP1 in the acute modulation of skeletal muscle protein synthesis during recovery from exercise. Fasting male rats were run on a treadmill for 2 h at 26 m/min and were realimented immediately after exercise with either saline, a carbohydrate-only meal, or a nutritionally complete meal (54.5% carbohydrate, 14% protein, and 31.5% fat). Exercised animals and nonexercised controls were studied 1 h postexercise. Muscle protein synthesis decreased 26% after exercise and was associated with a fourfold increase in the amount of eIF4E present in the inactive eIF4E.4E-BP1 complex and a concomitant 71% decrease in the association of eIF4E with eIF4G. Refeeding the complete meal, but not the carbohydrate meal, increased muscle protein synthesis equal to controls, despite similar plasma concentrations of insulin. Additionally, eIF4E.4E-BP1 association was inversely related and eIF4E.eIF4G association was positively correlated to muscle protein synthesis. This study demonstrates that recovery of muscle protein synthesis after exercise is related to the availability of eIF4E for 48S ribosomal complex formation, and post-exercise meal composition influences recovery via modulation of translation initiation.

Discussion: By analyzing the complex series of steps by which our muscle cells build new proteins we realize that there are many stages in this process where protein synthesis could be modulated. As you may know, protein synthesis declines at the onset of exercise and then resumes some time after exercise is finished. Unfortunately protein catabolism continues throughout exercise in order to liberate various amino acids for fuel and to act as intermediates in the Krebs cycle. Recovery from exercise begins with the stimulation of protein synthesis.

From the study above we see that it is translation inhibition that is responsible for the decline in protein synthesis rates during and after training. So what is the mechanism by which translation is inhibited by exercise? This is exactly what the researchers were trying to discover. Initiation of translation (the binding of mRNA to the ribosomal pre-initiation complex) requires group 4 eukaryotic initiation factors (eIFs). These initiation factors interact with the mRNA in such a way that makes translation (the construction of new proteins from the mRNA strand) possible. Two eIFs, called eIF4A and eIF4B, act in concert to unwind the mRNA strand. Another one called eIF4E binds to what is called the "cap region" and is important for controlling which mRNA strands are translated and also for stabilization of the mRNA strand. Finally, eIF4G is a large polypeptide that acts as a scaffold or framework around which all of these initiation factors and the mRNA and ribosome can be kept in place and proper orientation for translation.

Now it is eIF4E that appears to be a key point for modulation of translation, or protein synthesis. You see, eIF4E (at the mRNA cap) binds with eIf4G (scaffold) in order to form the functional complex (eIF4F) that allows translation of the mRNA. Some research shows that eIF4E activity is modulated by increased phosphorylation of the eIF4E molecule, which in turn increases its binding affinity for the mRNA cap region. This would effectively increase the amount of translation going on and ultimately the amount of protein synthesis. Another explanation involves a "binding protein" called 4E-BP1. It binds the eIF4E molecule making it unable to bind to eIF4G. This effectively would put a stop to the whole process.

As you read from the above summary, rats were exercised for two hours and then were withheld food, given only carbohydrates, or given a mixed meal composed of protein, carbs, and fat (Ensure powder). The results show us a few things.

1) Insulin alone is not enough to prevent 4E-BP1 from sequestering eIF4E. Remember eIF4E must be free to bind to eIF4G in order for protein synthesis to begin. Insulin as well as amino acids must be present at the same time as indicated by the results from the group that were fed a mixed nutrient meal. So although feeding of the carbohydrate meal resulted in elevated blood glucose and elevated insulin levels, it was not sufficient to allow protein synthesis to begin.

2) The only group that experienced a significant drop in cortisol levels was the mixed meal group. The carbohydrate only group showed that neither blood glucose nor insulin had any effect on reducing cortisol levels. In fact, the mixed meal group showed cortisol levels even below those in the control group who did no exercise and were also fed the same meal.

Although it is important to understand the mechanisms by which our bodies respond to exercise, understanding is not enough to build more muscle. You must put into practice what research shows us. In this case we learn that in order for protein synthesis to resume as soon as possible after training, we must consume an easily digestible meal containing at least both carbohydrate and protein. We also learn that elevated insulin as well as lowered cortisol levels are also necessary for protein synthesis to resume after training. Without a complete meal consumed right after your workout, key molecules needed for constructing proteins remain sequestered by binding proteins until your next meal. This is important because mRNA does not float around in your cell forever. It must be used quickly before it is naturally degraded by the cell.