November 1, 1998
Volume 1, Number 7
Research Update
by Bryan Haycock MSc., CSCS
bryan@thinkmuscle.com
Please send us your feedback
on this article.
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. 3rd ed. New York, W.H.
Freeeman and Company, 1986.
Starr C, Taggart R (eds): Biology: The unity and Diversity of Life. 6th
ed. Belmont,Ca., Wadsworth Publishing, 1992
Starr C, Taggart R (eds): Biology: The unity and Diversity of Life. 7th
ed. Belmont,Ca., Wadsworth Publishing, 1995
The first new anabolic drugs of the next millennium are discovered!
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.
Jump Starting Protein Synthesis After Exercise.
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.
by Bryan Haycock MSc., CSCS
bryan@thinkmuscle.com
Please send us your feedback
on this article.
