Thursday, June 6, 2013

Serum & Intramuscular Testosterone, DHT and Androgen Receptor Response to High vs. Low Volume Training

Another set for another ng of testosterone? Does it work that way and is it worth it - not just on paper, but in terms of real gains?
I know that we don't know! And among the many things we don't know the influence of the post-workout elevation in the long-thought "anabolic" hormones testosterone, growth hormone, and co. is unquestionably one of my personal favorites. You've read about it, here at the SuppVersity many times and I got to tell you in advance that the absence of convincing evidence for / against its importance will become a problem in the bottom line of today's SuppVersity article dealing with the intriguing results of an experiment that has been conducted by Lukas J. Farbiak as part of his Honors Thesis (Farbiak. 2013).

"Effects of Lower- and Higher-Volume Resistance Exercise on Serum Total and Free Testosterone, Skeletal Muscle Testosterone and Dihydrotestosterone Content, and Skeletal Muscle Androgen Receptor mRNA Expression and Protein Content"

That's quite a title for a thesis right? Well, one thing's for sure: Having the words, "high, "low", "training volume", "resistance exercise", "total and free testosterone", "dihydrotestostereone", etc. all in the headline is certainly an advantage when it comes to findability of a paper - or in this case - a thesis in a database. And in fact, it was really the title of the 91 page piece that has caught my eye, a couple of days ago - what peaked my interest, though were the research hypotheses Farbiak, whose thesis was by the way overseen by Darryn Willoughby, formulated:
  • H1 : Following the HV [high volume] exercise bout involving both upper- and lower-body resistance exercise, a significant increase in serum testosterone will occur compared to the LV [low volume] exercise bout only involving lower-body resistance exercise. 
  • H2 : Following the HV exercise bout involving both upper- and lower-body resistance exercise, a significant increase in muscle testosterone and DHT content will occur compared to the LV exercise bout only involving lower-body resistance exercise. 
  • H3 : Following the HV exercise bout involving both upper- and lower-body resistance exercise, a significant increase in AR mRNA expression and protein content will occur compared to the LV exercise bout only involving lower-body resistance exercise.
I took the liberty of highlighting three things in Farbiak's hypotheses, which tell you why you want to know the outcome of the study, even if the current "state of the research" questions the significance of exercise-induced elevations of androgens in terms of their ability to elicit muscle growth.

What's special, here, is that we are not measuring serum levels exclusively, but get a much more detailed picture of the endocrine response to high vs. medium volume training.

Why would the internal androgen levels differ from those outside of the cell? The notion that this could and in fact is the case did not arise before Hammes et al. discovered that contrary to the previously heralded position that says that only free testosterone levels would matter and that the latter would be able to enter the cells via passive diffusion, the entrance of testosterone into the cell is actually governed by (attention please) megalin, a low density lipoprotein receptor (LDR) related  protein. According to Hammes, SHBG can bind to megalin can internalize the SHBG + androgen pair into the cytoplasm, where the binding globulin is degraded and the steroid will be released to the cellular environment.It goes without saying that this changes the interpretation of previous data and provides a whole new perspective on the androgen - muscle interaction with the formerly "passive" bound testosterone suddenly having the ability to promote hypertrophy.
In this context the relation of free androgens, androgen receptor expression and the presence and concentration of intra-muscular may well provide first insights into why previous studies, which have predominantly relied on the determination of serum levels without even checking,
  • whether there were enough receptors to (this is an oversimplification) transduct the anabolic signal of workout induced increases in testosterone to the muscle cells, and
  • to which extent the changes in extra-cellular androgen levels correlate with the amount of testosterone and DHT that's actually in the muscle.
Now that I have your full attention let's take a look at what kind of workout program we are dealing with in the study at hand, for which the researchers recruited 10 "apparently healthy resistance trained  [regular,  consistent  resistance  training (i.e. thrice weekly) for  at least 1 year prior to the onset of the study], men between the ages of 18-30" (Farbiak. 2013).
"In a randomized, cross-over design, participants visited the laboratory on 5 separate occasions in the following manner: visit 1 = entry/familiarization session, visit 2 = testing/resistance exercise session 1, visit 3 = 24 hour follow-up for session 1, visit 4 = testing/resistance exercise session 2, visit 5 = 24 hour follow-up for session 2. Relative to the testing sessions (visits 2 & 4), participants performed a resistance exercise session involving the knee extension exercise on two occasions separated by one week. One session constituted the control session and was preceded by rest and the other was preceded by the experimental session and preceded by a bout of high-volume, moderate-intensity upper-body resistance exercise using short rest periods." (Farbiak. 2013).
The dependent variables, i.e. serum free and total testosterone, intra-muscular testosterone, DHT and  AR  receptor mRNA, as well as protein expression were determined on all, but the initial entry/familiarization visit.

The workout itself (remember this is not a chronic resistance training study, as the one by West et al. (2012) which is - at least to my knowledge unique wrt to the real-world relevance of the data; learn more) consisted of
  • LV - low volume: 5 sets of 5-RM (90%-95% 1-RM) of the bilateral knee extension exercise with 3 minutes of rest between sets.
  • HV - high volume: Upper-body resistance exercise protocol of 4 sets of 10-RM each of the bench press, seated row, and overhead shoulder press exercises immediately prior to the knee extension protocol
  • the initial load was set at 80% of the 1-RM for each participant. 
  • if muscle fatigue/failure occurred during a set, a spotter provided assistance until the participant completed the remaining repetitions and resistance was reduced for subsequent sets
In all cases, 2 minutes of rest separated sets and exercises. All training sessions were conducted in
the Baylor Laboratories for Exercise Science & Technology (BLEST) and supervised by study personnel.
Figure 1: Sum total and free testosterone in response to high and low volume training (Farbiak. 2013)
Now, the data in figure 1 actually mirrors what we already know: The overall serum response to high volume training is more pronounced that that to playing around on a leg extension machine (which happens to be the favorite benchmark for the / I repeat myself / likewise not very useful studies on PWO protein synthesis).
"Several studies have shown that acute resistance exercise bouts elicit a testosterone response (Kraemer. 1990; Kraemer, Gordon et al., 1991; Kraemer, Hakkinen et al., 1999; Spiering, Kraemer. 2008; Roberts. 2009). Such exercise bouts shown to elicit a testosterone response need to consist of a high intensity (load) (85%-95%) of one repetition max and meet a minimum threshold, and moderate to high volume (set x number of reps x intensity). Exercises that utilize large muscle groups (i.e. power clean, squats, and dead lifts) as well as performing exercises involving large muscle groups first, with short rest periods (30-60 sec) have shown to elicit the greatest response (Kraemer, Marchitelli et al., 1990; Spiering, Kraemer et al., 2008; Vingren, Kraemer et al., 2010). [...] It is known that the testosterone response resistance exercise is highly variable (Kraemer, 1988). Thus, it is possible that after multiple years of resistance training, the initial phasic response of the hypothalamus gonadal axis (aka. testosterone axis) response elicited by resistance exercise bout un trained individuals has become blunted from habitual resistance exercise. However, it is necessary that further research be conducted to elucidate why this blunted response occurs." (Farbiak. 2013; my emphasis of the key points)
As far as the differential response of free and total testosterone is concerned the tendency for both to go hand in hand has been observed in previous studies, as well (Durand. 2003; Kraemer. 1990; Kraemer. 1991; Kraemer. 1999; Spiering. 2008; Roberts. 2009). What's "new" or let's say something we have much less reliable data on are the changes that take place within the muscle (see figure 2)
Figure 2: Intra-muscular androgen & -receptor mRNA & protein expression (Farbiak. 2013)
Interestingly, enough those potentially far more relevant changes take place on a very different time-scale. While we do see the touted increases in serum testosterone in the immediate vicinity of the workout, the corresponding intra-muscular levels are actually declining from pre to post (red vs. blue bars). As Farbiak points out, these changes were yet statistically non-significant and to thus correspond to previous results presented by Vingren & Kraemer et  al. in 2008 (Kraemer. 2008). The same goes for the DHT response that did not make it past the p > 0.05 mark of statistical significance (FYI: this means the chance that this is just a statistical artifice is >5% and thus "not significant").

As far as the androgen receptor mRNA expression is concerned a often-cited (also by me, here at the SuppVersity) by Kraemer et al. observed a reduction in response to a single bout of resistance exercise, (Kraemer. 2010). The latter does actually conflict with in-vitro studies that suggested that the presence of higher testosterone levels would lead to an increased expression of androgen receptor mRNA and proteins - an observation of which Farbiak points out that it does not only stand in line with a previous study by Willoughby  and  Taylor (Willoughby. 2004) who observed a
"+35% and +43% increases in AR mRNA expression 48 hours after the first and third resistance exercise bouts, with a peak increase of 68% in AR mRNA expression occurring 48 hours after the second resistance exercise bout within the resistance exercise group" (Farbriak. 2013)
which was ascribed to corresponding increases in serum testosterone levels. In view of the fact that the latter were absent in Farbiak's subjects, it is not surprising that the existing increases in AR receptor mRNA in the study at hand did not reach statistical significance. Similarly, Farbiak was not able to show significant alterations in androgen receptor protein content in response to either LV or HV bouts of resistance exercise, which leaves us with pretty much of a null result and raises the question...

What do we make of this null result?

I guess the first thing would be to take a look at the underlying "mathematical" reason for the non-significance of the results... standard deviations - HUGE standard deviations, indeed. So huge that I initially thought that this must be a mistake, I mean if you have a mean pre-testosterone level of 43.59 ng/dl and a standard devition of 43.03 ng/dl, i.e. 99%, what can you expect? Now this is an extreme example, but in view of the relative small number of participants it should suffice to tell you that - maybe - we should not focus that much on statistical significance, here?

Suggested read: "Advanced Trainees Benefit from Increased Training Volume! Greater & Steadier Strength Gains with 8 Sets of Squats. Plus: Over 6 Weeks, 1 Set and 4 Sets Equally (In-)Effective." If higher volume begets higher T-responses and the latter is blunted in advanced trainees, it would appear logical that they benefit from doing more (learn more)
Schoenfeld mentioned similar effects in a whole host of pertintent studies in his excellent review of the literature on the effects of the exercise induced hormonal changes on muscle hypertrophy (I mentioned this review before, e.g. March 2, 2013; March 4, 2013). So it could simply be inter-individual variability that skewed the results. If that was the case, it is however unlikely to assume a dose-response relationship between any (serum or intramuscular) changes in androgens / androgen receptor expression and skeletal muscle hypertrophy - I mean that would imply much more pronounced differences in muscle growth in response to a workout than the real world results do indicate.

Another factor that may have influenced the results is the high training experience (>8 years) of the participants in the Farbiak study, if the initially cited hypothesis that the androgen response to exercise declines in experienced athletes turns out to be true, the non-significance of the endo- and paracrine hormonal response in the study at hand could well be "normal" and no anomaly. And if that was the case, it would suggest that the changes that were observed in previous studies, many of which were conducted on rookies, do matter - at least to a certain degree.

To use this as the only explanation for the (comparatively) exorbitant gains training noobs experience once they pick up their first dumb- and barbells would yet be shortsighted. To add it as yet one of the many confounding factors, on the other hand, would make perfect sense, as it would stand in line with the (comparably) short-term detrimental effects chronic resistance training without off-times has on the protein synthetic mTOR response to exercise (learn more about exercise induced "mTOR resistance").

Bottom line: To sum it up, while we do now have another puzzle piece, it looks as if it only made us realize that our 1,000 piece puzzle is in fact a 10,000 piece puzzle and that it will probably require more than just a handful of follow up study to investigate the numerous factors "such as age, time of day [not all trainees trained at the same time, so the circadian rhythm may be an issue, in the study at hand], and training experience" (Farbiak. 2013) of which Farbiak speculates in the discussion of his honors thesis that they may account for the observed discrepancies and inconsistencies in testosterone response to acute resistance exercise... ah, and once we've done that, we would need more studies like the one by West et al. (2012) to see the real world implications. I guess, we better issue a bond to get those finance, right?

References:
  • Farbiak, LJ. Effects of Lower- and Higher-Volume Resistance Exercise on Serum Total and Free Testosterone, Skeletal Muscle Testosterone and Dihydrotestosterone Content, and Skeletal Muscle Androgen Receptor mRNA Expression and Protein Content. A Thesis Submitted to the Faculty of Baylor University In Partial Fulfillment of the Requirements for the Honors Program. May 2013.
  • Durand RJ, Castracane VD, Hollander DB, Tryniecki JL, Bamman MM, O'Neal S, Hebert EP, Kraemer RR. Hormonal responses from concentric and eccentric muscle contractions. Med Sci Sports Exerc. 2003 Jun;35(6):937-43.
  • Hammes A, Andreassen TK, Spoelgen R, Raila J, Hubner N, Schulz H, Metzger J, Schweigert FJ, Luppa PB, Nykjaer A, Willnow TE. Role of endocytosis in cellular uptake of sex steroids. Cell. 2005 Sep 9;122(5):751-62. 
  • Kraemer WJ, Marchitelli L, Gordon SE, Harman E, Dziados JE, Mello R, Frykman P, McCurry D, Fleck SJ. Hormonal and growth factor responses to heavy resistance exercise protocols. J Appl Physiol. 1990 Oct;69(4):1442-50.
  • Kraemer WJ, Gordon SE, Fleck SJ, Marchitelli LJ, Mello R, Dziados JE, Friedl K, Harman E, Maresh C, Fry AC. Endogenous anabolic hormonal and growth factor responses to heavy resistance exercise in males and females. Int J Sports Med. 1991 Apr;12(2):228-35.
  • Kraemer WJ, Häkkinen K, Newton RU, Nindl BC, Volek JS, McCormick M, Gotshalk LA, Gordon SE, Fleck SJ, Campbell WW, Putukian M, Evans WJ. Effects of heavy-resistance training on hormonal response patterns in younger vs. older men. J Appl Physiol. 1999 Sep;87(3):982-92.
  • Kraemer WJ, Ratamess NA. Hormonal responses and adaptations to resistance exercise and training. Sports Med. 2005;35(4):339-61.
  • Roberts MD, Dalbo VJ, Hassell SE, Kerksick CM. The expression of androgen-regulated genes before and after a resistance exercise bout in younger and older men. J Strength Cond Res. 2009 Jul;23(4):1060-7. 
  • Schoenfeld BJ. Postexercise hypertrophic adaptations: a reexamination of the hormone hypothesis and its applicability to resistance training program design. J Strength Cond Res. 2013 Jun;27(6):1720-30.
  • Spiering BA, Kraemer WJ, Anderson JM, Armstrong LE, Nindl BC, Volek JS, Maresh CM. Resistance exercise biology: manipulation of resistance exercise programme variables determines the responses of cellular and molecular signalling pathways. Sports Med. 2008;38(7):527-40.
  • Spiering BA, Kraemer WJ, Vingren JL, Ratamess NA, Anderson JM, Armstrong LE, Nindl BC, Volek JS, Häkkinen K, Maresh CM. Elevated endogenous testosterone concentrations potentiate muscle androgen receptor responses to resistance exercise. J Steroid Biochem Mol Biol. 2009 Apr;114(3-5):195-9.
  • West DW, Phillips SM. Associations of exercise-induced hormone profiles and gains in strength and hypertrophy in a large cohort after weight training. Eur J Appl Physiol. 2012 Jul;112(7):2693-702.
  • Willoughby DS, Taylor L. Effects of sequential bouts of resistance exercise on androgen receptor expression. Med Sci Sports Exerc. 2004 Sep;36(9):1499-506.