IL-6 - True Muscle Builder or Just a Measure of Workout Intensity? Plus: If Testosterone Does Not Matter, Why Does the Androgen Receptor Density Make a Difference?

No matter how close we look, the influence of previous, i.e. GH, IGF-1 and testosterone, as well as novel, i.e. IL-6 and AR expression, potentially growth promoting suspects remains elusive.

SuppVersity readers know, there is more to "inflammation" than the average mass media article will make you believe. The same "bad cytokines" that will decrease your insulin sensitivity, make you sick and obese, when they leak from your "inflamed" beer belly, are actually the good guys, when they are released in response to a workout from the musculature. "Myo-", not "cytokines", that's how researchers refer to them (Pedersen. 2007); and their role in (exercise) metabolism and immunity is until now still not fully understood.

The results the scientists from the McMaster University probably won't add much to our understanding of the systemic effects of IL-6 and other myokines. What certainly do, though, is to support the notion that "inflammation" can have profound and very far-reaching effects on our physiology (learn more).

Does IL-6 build muscle?

The mere idea that IL-6, a molecule of which most people think that it was a good measure of how messed up your health actually is, could be a promoter, or at least a measure, of skeletal muscle growth in response to a workout sounds about as logical (or illogical, if you will) as the previously established fact that the allegedly anabolic hormones testosterone, IGF-1 and growth hormone don't show the slightest correlation with the exercise induced skeletal muscle growth (West. 2011; learn more in "Anabolic Workouts Revisited").

Figure 1: Relative increase in muscle CSA change for androgen receptor density and p70S6K expression (left) and individual correlation of endocrine changes and muscle CSA (right), data based on Mitchel. 2013)

What was to be expected, though, was the correlation between the skeletal muscle hypertrophy response to the 16 week / 4x per week exercise program the 23 previously untrained study participants had to endure, on the one hand, and the exercise induced increase in p70S6K, on the other hand. The signalling protein p70S6K is after all something like the "protein pump"-gauge in the mTOR cascade.

Strength training can increase the androgen receptor density

Less well-known, but also not really new is another observation Mitchel et al. made: The resistance training lead to an allegedly subject specific and overall insignificant increase in the density (number per area unit) of androgen receptors; and though the overall increase may not have been statistical significant, the correlation of the the increase in androgen receptor density and the lean muscle gains of the subjects was.

New myonuclei (blue) are necessary to keep growing (learn more)

What about the muscle structure? What I am missing in this study is a measure of the myonuclei and domain sizes. We know that inflammation plays a major role in the restructuring process of the musculature that's necessary to maintain myonuclear domain size and thus the capacity for muscle protein synthesis (Bamman. 2001). It does furthermore seem likely that the any increase in myonuclei number would go hand in hand with increases in the number of androgen receptors. The corresponding data could thus help us to answer some of the questions we still have about the immediate (mTOR) and the chronic (endocrine and immune) contributions to skeletal muscle hypertrophy.

Similar effects on the androgen receptor density have been described by Willoughby & Taylor in 2004, already. In the pertinent study from the Baylor University, this increase did yet go hand in hand with increases in total testosterone and the free androgen index, of which the authors say that they were brought about by persistent increases in testosterone - increases in testosterone that were not observed in the more recent study by Cameron Mitchel, where the testosterone levels dropped (albeit non significantly) in response to the 2x2 upper-/lower-body split training.

"Our study corroborates previous findings that mean AR protein expression was not increased following resistance training; however, the response had marked heterogeneity, with some subjects showing a marked (1.5 - 2.5 fold) increase in AR protein content [...] Despite no statistically significant change in AR receptor protein content, there was a correlation between AR protein content with fibre hypertrophy. Our results suggest that changes in AR content may be part of a muscle-specific response present to a greater degree in responders and responsible for some (~25%) of the variation in muscle fibre hypertrophy." (Mitchel. 2013)

With regard to the correlation (I want to emphasis that we don't have enough evidence to do anything, but speculate about causative effects, here), it may be worth mentioning that the statistical significant association between androgen receptor density and the changes in muscle cross-sectional area was more pronounced in the "strength type" fast twitch (type II) than in the "endurance type" slow twich muscle fibers (0.6, p = 0.002 vs. 0.47, p= 0.023).

For IL-6, on the other hand,  it was the exact opposite. Unlike the number of androgen receptors per muscle area, the amount of interleukin 6 that was released or, as Mitchel et al. say, "filtered" from the muscle into the blood stream in response to the workout exerted a minimally more pronounced effect on the slow twitch "endurance type" fibers (type II).

So is IL-6 anabolic? It is counter-intuitive and would be premature to say that "IL-6 plays a causal role in skeletal muscle hypertrophy". This is particularly true in view of the fact that high baseline IL-6 levels that would be indicative of chronic vs. acute inflammation were "inversely correlated with fibre hypertrophy" and that IL-6 per se "is associated with both muscle protein breakdown and JAK/STAT signalling in satellite cells" (Mitchel. 2013).

Acutely (green circle) IL-6 will help build muscle, chronically it will make you sick (Muñoz-Cánoves. 2013).

It does thus appear to be more likely to assume that the increased IL-6 response is - just as the previously observed increase in cortisol in West et al. (2011), by the way - a marker of the workout induced strain, which does - in a non-overtraining scenario! - predict the adaptive response and thus the actual muscle gains.

The fact that the inclusion of IL-6 in a model prediction of the exercise induced hypertrophy response did not increase its accuracy would also point towards a corollary, not a causal involvement of a cytokine, the local (=in the muscle) production of which has also been implicated in the protective effect of exercise against insulin resistance, as well as increases in lipolysis and fatty acid oxidation (Pedersen. 2007).

In the end I have to admit that the study at hand does not really offer the material that would be necessary to formulate something like practical implications. What the results Mitchel et al. present in their paper can do is to reaffirm that skeletal muscle hypertrophy occurs in a(n at least) bi-phasic process with (1) an acute "inflammatory" phase where P70S6K (part of the mTOR cascade) determines and IL-6 (and thus inflammation) correlates with an increase in lean muscle mass and (2) a long-lasting "recovery" phase where the increase expression of androgen receptors could play an important role in the maintenance of the immediate gains in skeletal muscle protein. Your training should thus provide for both: An intense hypertrophy stimulus on the training days and lots of time and nutrients to recover on your off days.

References: 

• Bamman MM, Shipp JR, Jiang J, Gower BA, Hunter GR, Goodman A, McLafferty CL Jr, Urban RJ. Mechanical load increases muscle IGF-I and androgen receptor mRNA concentrations in humans. Am J Physiol Endocrinol Metab. 2001 Mar;280(3):E383-90
• McKay BR, De Lisio M, Johnston AP, O'Reilly CE, Phillips SM, Tarnopolsky MA, Parise G. Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans. PLoS One. 2009 Jun 24;4(6):e6027. 
• Mitchell CJ, Churchward-Venne TA, Bellamy L, Parise G, Baker SK, et al. Muscular and Systemic Correlates of Resistance TrainingInduced Muscle Hypertrophy. PLoS ONE. 2013; 8(10): e78636
• Muñoz-Cánoves P, Scheele C, Pedersen BK, Serrano AL. Interleukin-6 myokine signaling in skeletal muscle: a double-edged sword? FEBS J. 2013 Sep;280(17):4131-48. doi: 10.1111/febs.12338. Epub 2013 Jun 18.
• Pedersen BK, Akerström TC, Nielsen AR, Fischer CP. Role of myokines in exercise and metabolism. J Appl Physiol (1985). 2007 Sep;103(3):1093-8. Epub 2007 Mar 8. Review.
• 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. doi: 10.1007/s00421-011-2246-z. Epub 2011 Nov 22.
• 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.

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.

Carnitine as Repartitioning Agent? IGF-1, p-AKT & mTOR Up, Catabolic Proteins Down + 7% Improvement in Lean- to Total Mass Ratio W/ HED of 1-1.5 of Carnitine/Day

It won't spare you the sweat, but carnitine could make it even more worthwhile by ramping up the anabolic and shutting down the catabolic signals.

Until 2006 l-carnitine has been known as a fat-burner, an in-effective fat-burner and an expensive and pretty useless supplement (depending on whom you were asking). Then, in July 2006, Kraemer et al. published a paper (a human study, above all!) in the journal Medicine & Science in Sports and Exercise a consequential paper so to say; a paper in which the authors report that l-carnitine l-tartrate supplementation at a dosage of 2.933g/day (this amount of LCLT contains 2g of pure carnitine) led to a statistically significant increase in androgen receptors in the vastus lateralis after a heavy resistance training protocol in previously strength trained male subjects (Kraemer. 2006).

Still, the evidence has always been inconclusive to say the least

Despite the fact that the concomitantly elevated post-workout luteinizing hormone levels (+19%) Kreamer et al. observed would tell you that the testosterone that would have been necessary to activate those receptors was already on its way, I have never considered this study as convincing evidence of the anabolic prowess of l-carnitine. Plus, let's be honest, differences in whatever serum markers in response to an acute bout of resistance training have failed us way too often, not to look at studies like these with appropriate skepticism.

Do you remember the Ratames study from 2005? The one that showed that high volume training lowers the no. of androgen receptors on the trained muscles? This certainly makes l-carnitine sound like the perfect addition to high volume routines, right? (learn more)

That the same principle of "calm down and don't get too excited over the results of a single trial" does all the more apply to rodent studies should be self-evident and still, science is all about taking each and every experimental result into account to form a theory that can explain all of them, or, alternatively, is able to bust short-comings in previous studies that don't comply with the predictions of the respective theory.

Now, the soon-to-be-published paper by Janine Keller and her colleagues from the University of Giessen (Germany) certainly qualifies as part of the evidence we simply cannot ignore, when we are looking for evidence in support of the theory that l-carnitine could be an overlooked muscle builder or repartitioning agent.

After all, their observation of decreased levels of the proteolytic (=catabolic) MuRF1 protein, as well as the ubiquitin-protein conjugates, which are increased in catabolic states such as starvation and atrophy denervation (cf. Wing. 1995) , alone, would signify that l-carnitine could make a valuable addition to everybody's supplementation regimen.

Lower catabolism + increased anabolism = ???

There is more, however, the addition of 1250 mg L-carnitine/kg to a basally "low carnitine" vegetarian diet also led to significant increases in systemic IGF-1 concentrations in plasma and a local increase in the activity of the PI3K/Akt/FoXO-1 signalling pathway (see figure 1)

Figure 1: IGF-1 mRNA and serum levels, as well as the muscle specific expression and phosphorylation (ph) Akt, mTOR & co after four weeks on the low or high carnitine diets (Keller. 2013)

These results do yet not stand in isolation as the ones by Kraemer et al. still do. Other recent studies by the same research group in Giessen, as well as colleagues from the University of Barcelona have already confirmed the anti-catabolic effects of l-carnitine in piglets and a cancer cachexia model in rodents, respectively (Keller. 2012; Busquets. 2012).

"And you are telling me that works in humans, as well? "

What's the best form of carnitine to take to elicit these effects: I knew you would ask this, so I react to two facebook questions by adding this red box willingly admitting that I just cannot tell you what the best form of carnitine is. There simply is no study that would compare e.g. acetyl-l-carnitine (ALCAR) and l-carnitine l-tartrate (LCLT) in a scenario that would be relevant to the above question. What I can tell you though, is that it appears as if you were better off with LCLT than with ALCAR, if your goal is to top off your intra-muscular carnitine levels. That being said, even normal creatine can do that - you will just have to take more of it. If you are looking for more information you can check out the part of the Amino Acids for Super Humans Series that's dealing with "the carnitines", here.

In this context it does yet also have to be mentioned that the effects of l-carnitine are at least in part species specific. How we know that? Well, in contrast to the said study by Basquets et al. the provision of an carnitine to piglets (Keller. 2012) did not only reduce the MuRF-1 expression, but also the level of its likewise catabolic E3 ligase cousin atrogin-1.

"It has been shown that myofibrillar proteins, like myosin light chain proteins are the main targets of MuRF1for ubiquitination. Thus, carnitine might suppress particularly the degradation of myofibrillar proteins, which under physiological conditions comprise around 60% of total muscle proteins. In contrast to MuRF1, atrogin-1 tags primarily proteins for degradation which are important for controlling protein synthesis and myoblast differentiation, like myogenic factor MyoD, myogenin and the eukaryotic initiation factor of protein synthesis eIF3-f." (Keller. 2013)

With pigs usually being a superior model of the human physiology, this would suggest that the anti-catabolic effects l-carnitine could have on humans are probably more, not less pronounced than those that were observed in previous rodent studies.

Whether the same goes for the IGF-1 response cannot be said, but just like the anticatabolic effects, the pro-anabolic increase in IGF-1 has been observed in previous trials, including a human trial by Di Marzio et al. who observed a significant increase in IGF-1 in HIV patients in response to the provision of 3g/day of acetyl-l-carnitine (Di Marzio. 1999). In the absence of the existing evidence from animal studies, these results would yet have little significance for healthy human beings, whose growth hormone and IGF-1 levels are not rock bottom to begin with (Viganò. 2003).

---

Bottom line: Irrespective of the absence of human data on the IGF-1 boosting effects from non-HIV patients - or even better in training scenarios - it would warrant future studies if an adequate amount of carnitine in the diet can exert beneficial effects in non-obese human beings. For the "sedentary", or let's rather say non-exercised rodents in the study at hand, the latter was a mere fat loss effect - despite the elevations in p-AKT, m-TOR, IGF-1 and the overall more "anabolic" state the rodents were in their lean body mass was not increased compared to their peers on the low carnitine diet.

"Just another set!" ... "I don't know man, we've already pumped away 100,000kg today... do you really believe that's productive, I mean, yeah, we are cuttin', but still" ...learn what this dialog is all about and whether and if / when "another set" is / isn't a good idea (read more)

The lean-to-total mass ratio of the rodents, on the other hand was ~7% higher in the rodents in the high carnitine group. If we do however take into consideration that most of you will not be vegetarians and thus not similarly carnitine deprived as the rodents in the control group on the <1mg/kg carnitine diets, it is highly questionable if the addition of the human equivalent of the 1.25g/kg chow, i.e. 15mg/kg body weight (HED) would actually yield any measurable benefit to non-vegetarians - irrespective of whether they train or not. After all, even the average omnivore human being consumes 100-300mg of carnitine per day (Broquist. 1994), so that the difference between your basal carnitine intake and the supplemental equivalent dose of 1050-1500mg/day is more than 100x lower than the exorbitant difference between the low (if not deficient) carnitine diet in Keller's rodent study at hand (remember: the basal diet had less than 1mg/kg chow; the supplemented diet hat 1250mg/kg diet!).

So what's the verdict then? I guess, I will leave the final words to Burke et al. who reviewed the usefulness of carnitine as an ergogenic aid in one of the first installments of the "A-Z Supplement Review" in the British Journal of Sports Medicine and wrote "future work with l-carnitine may also find some useful outcomes" (Burke. 2009) - needless, to say that the SuppVersity is going to be the place, where you will read about it first ;-)


References:

• Broquist HP. Carnitine. In Shils ME, Olson JA, Shike M (eds): "Modern Nutrition in Health and Disease." Malvern, PA: Lea & Febiger, 1994. 459– 465.
• Burke LM, Castell LM, Stear SJ, Rogers PJ, Blomstrand E, Gurr S, Mitchell N, Stephens FB, Greenhaff PL. BJSM reviews: A-Z of nutritional supplements: dietary supplements, sports nutrition foods and ergogenic aids for health and performance Part 4. Br J Sports Med. 2009 Dec;43(14):1088-90.
• Busquets S, Serpe R, Toledo M, Betancourt A, Marmonti E, Orpí M, Pin F, Capdevila E, Madeddu C, López-Soriano FJ, Mantovani G, Macciò A, Argilés JM:  l-Carnitine: An adequate supplement for a multi-targeted anti-wasting therapy in cancer.  Clin Nutr. 2012;31:889–895.
• Di Marzio L, Moretti S, D'Alò S, Zazzeroni F, Marcellini S, Smacchia C, Alesse E, Cifone MG, De Simone C. Acetyl-L-carnitine administration increases insulin-like growth factor 1 levels in asymptomatic HIV-1-infected subjects: correlation with its suppressive effect on lymphocyte apoptosis and ceramide generation. Clin Immunol. 1999 Jul;92(1):103-10.
• Glass DJ:  Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol. 2003; 5:87–90 .
• Kraemer WJ, Spiering BA, Volek JS, Ratamess NA, Sharman MJ, Rubin MR, French DN, Silvestre R, Hatfield DL, Van Heest JL, Vingren JL, Judelson DA, Deschenes MR, Maresh CM. Androgenic responses to resistance exercise: effects of feeding and L-carnitine. Med Sci Sports Exerc. 2006 Jul;38(7):1288-96.
• Keller J, Ringseis R, Koc A, Lukas I, Kluge H, Eder K:  Supplementation with l-carnitine downregulates genes of the ubiquitin proteasome system in the skeletal muscle and liver of piglets. Animal. 2012;6:70–78.  
• Keller J, Couturie A, Haferkamp M, Most E, Eder K. Supplementation of carnitine leads to an activation of the IGF-1/PI3K/Akt signalling pathway and down regulates the E3 ligase MuRF1 in skeletal muscle of rats. Nutrition & Metabolism. 2013; 10:28. 
• Lösel D, Rehfeldt C. Effects of l-carnitine supplementation to suckling piglets on carcass and meat quality at market age. Animal. 2013 Mar 11:1-8.
• Salama AF, Kasem SM, Tousson E, Elsisy MK. Protective role of L-carnitine and vitamin E on the testis of atherosclerotic rats. Toxicol Ind Health. 2013 Feb 13.
• Viganò A, Mora S, Brambilla P, Schneider L, Merlo M, Monti LD, Manzoni P. Impaired growth hormone secretion correlates with visceral adiposity in highly active antiretroviral treated HIV-infected adolescents. AIDS. 2003 Jul 4;17(10):1435-41.
• Wing SS, Haas AL, Goldberg AL. Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation. Biochem J. 1995 May 1;307 ( Pt 3):639-45.

Intermittent Thoughts on Building Muscle: Understanding the "Big T" - Testosterone Programs Stem Cells to Become Muscle not Fat + Keeps Satellite Cells & Motoneurons Alive

Image 1: Graphical summary of the probably best known function of testosterone - including who are not so "profane" as building muscle and getting ripped ;-)

In the last two installments of the Intermittent Thoughts, I have tried to convey a realistic perception of what exactly the effects of both supra- (that is below) and super- (that is above) physiological (that is "normal" in the sense that they represent the "average" male human being) levels of testosterone on body composition are. In this installment of the series I am now going to provide more information on the "exact" molecular underpinnings by which testosterone works its muscle building and fat burning magic. There is however one thing related to data I presented in the previous installments, I want to emphasize again: The use of a testosterone enanthate in the Bhasin study makes it very difficult to use the data to make prognoses with regard to the results you would see, when you use natural (or unnatural) supplements to raise the endogenous (produced by your testes) production of testosterone. And although there are certainly dozens of factors that would preclude respective inferences, I am going to address only those three, of which I believe that they are the most significant ones.

Three things to keep in mind, when you interpret the data from the last installment(s):

1. With testosterone enanthate having a ~4-5 day half-life, the testosterone levels, which, in the Bhasin study, were measured on day 7 after the injection, represent only a <50% remainder of the testosterone levels we would see within 24 hours post injection.
   

   

   Figure 1: Hypothetical serum testosterone levels in the course of the first seven days after the injection of endogenous testosterone (blue) compared to the regular diurnal rhythm (green) and the levels in response to a pretty potent (+70%) natural testosterone booster (red; all data has illustrative value, only)

   In spite of the fact that the data in figure 1 is obviously not based on "real" experimental data, I hope that by taking a brief look at the ratios of the areas under the curve of the
   
   
   • "normal" testosterone level with its ~40% daily variation (green), the...
      
   • +70% (maximally) naturally boosted testosterone level (red) and a ...
      
   • testosterone enanthate injection (blue),
      
   all of you will understand why the "muscle building / fat burning" effects of a +70% boost in testosterone from whatever OTC product you may be taking can hardly compare to injectable testosterone.
    
2. Another aspect that should be taken into account is the non-existent sex hormone binding globulin (SHBG) response in the Bhasin study, due to which the relative increases in bound and free (=unbound and purportedly "active") testosterone were identical. This can, but does not necessarily have to be the case, when you raise your testosterone levels "naturally". In that, the aromatization of testosterone to estrogen, appears to be one of the major correlates (I am deliberately not speaking of "causation" in this context) of increases in SHBG. In the worst case, you could thusly "boost" your total testosterone and end up with less free test due to a (possibly estrogen induced / related) increase in SHBG. That being said, I know a hand full of cases, where the exact opposite is the case. Especially very lean (yet still muscular) men tend to have low SHBG levels, so that despite "low-normal" total testosterone many of them have normal-high or even very high free testosterone levels.
    
3. The last factor that makes a direct quantitative comparison of the effects "naturally" and "artificially" elevated testosterone levels questionable, to say the least, is the absence of the natural diurnal rhythm with exogenous testosterone administration. In the course of 24h the testosterone levels fluctuate by +/-40% with a spike in the morning (around 6-7am) and a trough in the early evening. Contrary to the "artificially enhanced" testosterone levels, the ones on the printout from your lab thusly represent either the daily max (if the blood was drawn early in the morning), an average (blood drawn around noon) or the nadir (blood drawn in the evening) of your 24h testosterone level.
   Just as an aside: Imagine you wanted to sell a "natural test booster". What would be the best way to get a "clinically proven" rise in testosterone? Right! You just get your "study" participants tested in the evening for baseline and in the morning for post-intervention levels and *bang* you got your "clinically proven" +40% increase in testosterone ;-)
   And even if you managed (by whatever means) to "naturally" raise your testosterone to a level that you would "on average" have +200% the natural negative feedback mechanism (inhibition of luteinizing hormone (LH) release) will soon put an end to your thusly short-dated testosterone boost.

All that does yet not change the observation we have made in the first installment of this (hitherto) three-part series about the effects of testosterone on skeletal muscle hypertrophy: Testosterone builds muscle! The underlying physiological processes, however, are not fully elucidated. The brief summary I have put together in the following paragraphs is thusly a "work in progress" not only because I am still trying to figure out "how testosterone works", but also because the complex interplay of hormones, protein signalling cascades and key players of the immune system simply has not been fully elucidated, yet.

Direct effects of testosterone on muscle cells

I don't know if you have ever heard the name "Vida", if not, then you have probably not delved into the depth of bro-scientific steriodology. Julius A. Vida's book Androgens and Anabolic Agents was published in 1969 is what some people would call the "steroid bible". It contains information about the structure and biological activity of 666 different steroids.

Figure 2: Scan from Vida's book showing data on the androgenic and anabolic activity of 19-Nortestosterone (Nandronole, aka DECA) from a rodent model.

With the latter being of particular interest for roid / pro-steroid producers and consumers, scans of the tables, that make up a good part of the original book can be found on bulletin boards all over the Internet (cf. figure 2). Vida obtained the data from rodent studies and estimated the "anabolic" effect of the tested compounds based on the hypertrophy response of the levator ani muscle of his lab animals. Now, you may rightly ask yourself, how that relates to the topic at hand... well, the reason Vida (and most other researchers) chose the levator ani muscle as a benchmark is its high responsiveness to androgens, because it has a much greater androgen receptor (AR) density than the most of the skeletal muscle you are probably trying to build, when you are at the gym (well, I assume you don't train the levator ani, do you? ;-).

Image 2: The levator ani muscle is especially prone to androgen induced hypertrophy, because it has a particularly high amount of androgen receptors. Whether this is something you are particularly happy about or not, does not matter, in 99% of the cases that you read about the "anabolic activity" of a given "designer steroid", the latter is usually provided relative to the testosterone-induced hypertophy response of this muscle.

Interestingly, the areas of the muscle with the highest androgen receptor expression are the myonuclei and the satellite cells. You know both of them from previous installments of this series and will certainly remember that the recruitement of new myonuclei from satellite cells was a necessary prerequisite for continuous muscle growth, because with ever-increasing myonuclear domain sizes, the muscle will eventually become disfuctional (cf. "Growing Beyond Limits"). It is thusly likely to assume that, next to IGF-1, testosterone provides a second, secondary or complementary growth stimulus to the otherwise quiescent satellite cells. From the fact that the subjects in the Bhasin study exhibited a marked hypertrophy response in the absence of adequate training stimuli, we may also further conclude that the action of testosterone, contrary to the previously discussed locally expressed IGF-1 splice variants (cf. MGF & Co), does (at least up to a certain degree) not depend on muscle damage / strength training. The results of a 2005 study from the Human Performance Laboratory at the University of Connecticut (Kraemer. 2005), which found a -46% reduction in androgen receptor expression in response to volume (not single set, though) training, would even suggest, that testosterone takes a backseat, whenever the MGF-pathway is doing its muscle building job.

Whether the latter, i.e. testosterone's job in building muscle, is identical to the one of IGF-1 and its splice variants is debatable, anyways. After all experiments with isolated bovine satellite cells have shown that incubation with the synthetic androgen trenbolone lead to dose-dependent increases in protein synthesis and decreases in protein degradation (Kamango-Sollo. 2011). The function of testosterone could thusly be to maintain myoblasts (=progenitor cells) in the proliferate state - or, put more simply, testosterone keeps the satellite cells alive and ready to be incorporated into the muscle, whenever this becomes necessary.

Testosterone turns potential fat into muscle

Despite the fact that the muscle building effects of testosterone are at the heart of this series, I guess that you were similarly impressed by the effect the administration of graded doses of testosterone enanthate had on the body fat levels of the subjects in the Bhasin study. One possible explanation for this effect would certainly be the increased energy demands of the additional skeletal muscle mass. This alone can however hardly explain the profundity of the negative effects Bhasin et al. observed in the low and very low dose testosterone enanthate groups.

Figure 3: Relative change in lean and fat mass in response to changes in serum testosterone levels; the green area indicates "normal" = physiological testosterone levels; the asterisks (*) denote statistically significant (p < 0.05) changes vs. baseline (calculated based on Bhasin. 2001)

I mean, if you take a close look at the data, even the low-dose groups effectively gained some muscle mass (<2% and statistically non-significant). A loss of skeletal muscle mass thusly cannot explain the 18-37% increase in fat mass (cf. figure 3). In a subsequent publication Bhasin et al. thusly propose a - I may say quite exciting - alternative explanation for this and similar observations in hypogonadal men (Bhasin. 2004):

[...the] reciprocal change in lean and fat mass induced by androgens is best explained by the hypothesis that androgens promote the commitment of mesenchymal pluripotent cells into myogenic lineage and inhibit adipogenesis through an androgen receptor mediated pathway.

This priming effect testosterone has on the "universal" stem cells from connective tissue would not only result in a greater amount of stem cells that are to become muscle cells (in other words: satellite cells), testosterone would also reduce the amount of "future adipocytes" and thusly inhibit the formation of new and the replenishment of apoptotic, i.e. dead, fat cells. This hypothesis is corroborated by  recent findings of Semirale et al. who report that reduced visceral and subcutaneous fat accumulation with a reciprocal increase in lean mass in male mice with targeted androgen receptor over-expression in mesenchymal stem cells (Semirale. 2011).

The role of testosterone in the mind-muscle connection

Its effect on the actual muscle cells and their progenitors aside, testosterone also binds to the androgen receptors on the motoneurons that innervate the muscle. Interestingly, the death of these motoneurons, is considered the primary cause for sarcopenia and the associated decrease in muscle mass in the course of the aging process (Narici. 2008). Direct treatment of motoneurons with different doses of testosterone leads to increases in motoneuron size and number (Fraley. 2002; Mansouri. 2003). The physiological equivalent of the latter may thusly well be responsible for the improvements in the "mind-muscle connection" users of performance enhancing drugs frequently report. It may also facilitate a greater / optimized activation of existing muscle fibers and could thusly contribute to strength gains which would not depend on previous muscle growth. The increase in strength, in turn, would allow athletes to lift heavier weight and provide a novel growth stimulus, and so on...

Whenever there is talk of androgens and the "mind-muscle connection", someone usually mentions the three letters D, H and T and thusly invokes the role of the most potent androgen, dihydrotestosterone, to which the "Big T" is nothing but a prohormone. Whether it really is DHT, a combination of both, or if one is just more potent in inducing these androgen-related neuronal effects, will however be a topic for the next installment of this series,  in which DHT and estrogen will round out a still very sketchy portray of the complex role the "sex hormones" play in an orchestrate that is so complex that the notion that one hormone, protein, amino acid, or inflammatory cytokine alone could make your muscle grow is simply ridicolous - even if this hormone is "The Big T" ;-)