by Alfredo Franco-Obregón, PhD
Background
Many commercially available nutritional supplements are marketed on the promise that they will potentiate muscle development in response to exercise. That is, many manufacturers would have you believe that your efforts in the gym will be rewarded sooner by taking their products…. However, when examined in controlled scientific studies, most of these putative anabolic agents fail to promote muscle growth.
Much of the muscle growth that ensues with training arises from the re-initiation of the developmental program that originally ran during fetal development, a process known as myogenesis. Specifically, myogenesis consists of two phases: First, previously dormant muscle stem cells awaken from stasis and start to divide, greatly increasing their numbers. These activated stem cells are now known as
myoblasts. Next, myoblasts fuse with existing muscle fibers to replace damaged muscle tissue as well as to add mass and increase overall size to the existing muscle.
The outcome of myogenesis is subtly different when examined in the laboratory; that is, under artificial conditions using a technology known as tissue culture. When myoblasts are removed from an animal and grown in plastic
tissue culture dishes devoid of adult muscle fibers, myoblasts instead first fuse with each other to form elongated myotubes (see figure below). Subsequently, after the first
myotubes have been successfully established in the dish, myoblasts fuse with these as well.
![[IMG]](http://www.creatinemonohydrate.net/images/myoblasts.jpg)
Top: Individual muscle stem cells (myoblasts) aligning themselves into linear arrays in preparation for cell fusion to form myotubes, the first stage in the formation of an muscle fiber.
Bottom: Close-up of myotubes arising from the fusion of myoblasts as shown above.
So, given that myoblasts fuse with myoblasts in tissue culture, is examining myogenesis in dishes (in vitro) irrelevant? Most experts agree that myotubes are good approximations to the earliest skeletal muscle fibers produced in an animal and thus, can be taken as an indication of efficacious muscle development. Regardless, it remains unclear whether myotubes are produced at all in humans undertaking training, or if most of the new muscle that results from exercise is a consequence of myoblasts fusing with preexisting muscle fibers – a very important distinction for those working in the fields of muscle and exercise physiology. The production of new myotubes (inside the animal) would give rise to a process known as hyperplasia, whereas myoblasts fusing with preexisting muscle fibers would increase muscle size via a mechanism known as hypertrophy - two fundamentally distinct processes.
The study we are focusing on today was an initial attempt to quantify the direct myogenic effects of several popular ergogenic agents in vitro. Direct..., since muscle cells grown in tissue culture are not exposed to the hormonal environment present within the animal as well as are not subject to the mechanical forces provoked by the nervous system in initiating movement. In other words, any change in myogenic capacity measured in tissue culture would have to be a direct effect of the ergogenic agent in question and not a downstream consequence of the effect that exercise has on anabolic hormone balance - an indirect consequence of creatine supplementation.
Description of Study
This study tested the effects of several reputed ergogenic agents including two forms of creatine (monohydrate and pyruvate), an amino acid (L-glutamine), two steroidal agents (dehydroepiandrosterone (DHEA) and androstenedione) and two herbal extracts, (Ma Huang (Ephedra sinensis) and Zhi Shi (Citrus aurantium)) on muscle development in vitro.
The results of this study can be broken down to the effects that each ergogenic agent had over three key aspects of myogenesis: 1) alterations in muscle stem cell (myoblast) proliferation capacity; 2) changes in the ability of myoblasts to form myotubes and;
the influence of insulin over myogensis.
1) Creatine monohydrate generally did not interfere with the ability of myoblasts to divide and increase in number. Surprisingly, creatine pyruvate more consistently slowed the rate of cell division. On the other hand, androstenedione and DHEA killed myoblasts at a moderate concentration (micromolar) range, whereas Ma Huang and Zhi Shi proved lethal to myoblasts at all concentrations tested.
In summary, creatine monohydrate was the most permissive for myoblasts proliferation of all the agents testeds.
2)
Only creatine monohydrate had a positive effect over the ability of myoblasts to produce myotubes. Interestingly, the lowest dose of creatine monohydrate tested turned out to be the most efficacious at promoting myotube formation. Specifically, 0.1% (grams/liter) creatine monohydrate stimulated myotube formation, whereas 0.25%, 0.50% or 1.0% creatine monohydrate actually inhibited the formation of myotubes formation as well as slowed the proliferation of myoblasts. This result implies that overdosing with creatine may be counterproductive to muscle growth (resulting from myoblast fusion). This may be a simplistic interpretation of the data, however, as other anabolic attributes of creatine monohydrate (in conjunction with exercise) may compensate for this possible inhibitory effect.
Muscle cells in tissue culture must be supplied with a nutrient source (media) containing glucose, amino acids, and animal serum. Moreover, serum is an essential source of metabolites, hormones, currently unidentified nutrients and growth factors that permit the muscle cells to survive outside of the animal. All of the agents tested in this study were added directly to cell media containing serum, which could complicate the analysis of the results.
Given the success of the preliminary tests with creatine monohydrate in serum-containing media, the authors of the study next sought to identify any component of animal serum that may act in conjunction with creatine to promote muscle development. The two obvious candidates were insulin and the insulin-like growth factors – known myogenic agents. This group of researchers thus next examined the combined effects of creatine monohydrate and a special (serum-less) media formulation supplemented with insulin. The combination of insulin and creatine monohydrate was better at inducing myotube formation than the defined media containing insulin. On the other hand, somewhat surprising was the finding that creatine monohydrate without insulin was less effective than insulin alone at stimulating myotube formation, suggesting that the myogenic benefit of creatine monohydrate is strongly potentiated by insulin (or the insulin-like growth factors).
Creatine: A practical guide discusses how creatine monohydrate and the insulin-like growth factors act synergistically to promote muscle development –
independently of exercise!
A Potential Criticism of the Study
Sheep myobasts were used, as they were readily available to the authors of the study. Therefore, for the sake of relevancy, these same agents should also be tested on myoblasts isolated from human muscle.
Take Home
These preliminary results would suggest that creatine monohydrate directly promotes the formation of immature muscle fibers. Oddly, creatine pyruvate instead seemed to compromise myoblast survival and undermine myotube formation. Exactly why creatine monohydrate exhibits this mysterious effect is currently not fully understood. Some hints as to what aspects of creatine monohydrate may contribute to its unique pro-myogenic effect, however, are provided in past issues of the Creatine Newsletter.
Interestingly, the other commonly used ergogenic agents, L-glutamine, DHEA, androstenedione, Ma Huang and Zhi Shi, had either no effect, or actually killed myoblasts, before they could produce myotubes - an anti-myogenic effect.
This provocative pilot study certainly merits a follow up...
Scientific References
(Ref. 1) Vierck, J. L. et al. (200
The effects of ergogenic compounds on myogenic satellite cells. Medicine & Science in Sports & Exercise, Volume 35 (5), pages 769-776.
--------------------------------------------------------------------------------
Creatine Newsletter #35: What is the best creatine for muscle development?
