When Rodents Squat, Scientists Gain Insights into How Muscles Grow. IGF-1 Response to Exercise Does Matter - Locally, not Systemically, of Course!

You want to build big wheels? Look no further get yourself the "Squat T-Bar" with integrated 15mA electrical 'motivator' (Aguiar. 2013)

"A rodent study investigating strength workouts?" Yeah, I know it does not sound like that would be in any ways news-worthy, but if you take a look at the image on the right, you will immediately realize: This study is different! Instead of using a treadmill or simply stitching down (or rather up) one of the hindlimbs of the rodents to induce a chronic overload on the other one (don't laugh, many rodent studies have done just that), the study at hand (Aguiar. 2013), which is going to be published in the next issue of the International Journal of Sports Medicine, used a not innovative, but unfortunately largely forgotten (or overlooked?) torturing device that has been developed by Japanese researchers roughly 20 years a ago (Tamaki. 2013).

The rodent torture... ah pardon squat rack ;-)

After being fitted with a canvas jacket in a way that would enable the researchers to limit the twisting and flexion of their torsos (no, that was not a weight lifting belt ;-), the 32 male Wistar rats (80 days old, 250–300 g) were suspended in a standard position on their hind limbs and "encouraged" to exercise by "electrical stimulation [...] that was applied to the rat’s tail through a surface electrode"  (Aguiar. 2013).

Using their neat little toy, the eight researchers from the University Estadual Paulista, in Botucatu, Brazil, were able to submit the rats to a relatively realistic progressive resistance training regimen for either 8 or 12 weeks. Three times per week each rodent had to do 4 sets  of squats for 10-12 repetitions at 65-75% of its individual 1-RM (maximal weight the rodent could handle). During the study period, Aguiar et al. adjusted the weights twice a week to ensure the same training intensity throughout the experiment (something I would highly recommend to anyone of you, as well; try to pack on 1.25lbs - 2.5lbs at least every other week).

Figure 1: Body weight, muscle weight (plantaris, only) and food intake relative to body weight of the control (C8, C12) and trained (T8, T12) rats before and after the 8-week (C8, T8) and 12-week (C12, T12) intervention (data adapted from Aguiar. 2013)

As you can see in figure 1, this minimalist approach to leg training lead to an increase in both body weight and muscle weight that may initially look as if it was strongly linear. You do yet have to be careful about statements like that, because (a) the rodents did gain weight irrespective of whether they were training or not (80 day old rats are still growing!), so comparing the four bars next to each other and saying "yep, linear!" is not feasible, because this would mean linear as in not training for eight weeks < training for 8 weeks < not training for 12 weeks < training for 12 weeks, which is obviously nonsensical. That being said, there is simply (b) insufficient data to say anything about the linearity -- after all, we do have only three data points per group.

"All groups started the experiment with similar body weight. There was a significant increase (p < 0.05) in the body weight of the 4 groups in the resistance training program (C8: 35.5 %; T8: 27.7 %; C12: 46.9 %; and T12: 40.1 %) and final body weights were not significantly (p > 0.05) different between groups. Furthermore, no significant (p > 0.05) differences in the weekly food intakes were observed between the groups."(Aguiar. 2013)

What does yet stick out, is that the obviously age-dependent weight gain in the control groups C8 and C12 did not increase the weight of the plantaris muscle to a weight anywhere near to the muscle weight, the rats in the trained groups achieved.

Muscle gains and strength gains went hand in hand

In the rats who were subject to the three-times-per-week exercise regimen, on the other hand, those increases in muscle size went hand in hand with highly significant improvements in 1-RM squat power; While all groups had begun the training protocol with similar absolute 1-RMs of ~450g (that's about 130% body weight, pre) ...

"[...] training for 8 and 12 weeks promoted a significant (p < 0.05) increase in the RM/BW ratio in the T8 (pre- vs. post-training: 35.7 % increase, p < 0.05) and T12 (pre- vs. post-training: 57.1 % increase, p < 0.05) groups, while no statistical (p > 0.05) difference was observed in their respective control groups." (Aguiar. 2013)

Consequently, the ratio of 1-RM to body weight was 36.1 % and 57.7 % higher in the groups who had been training fot the last 8 or 12 weeks than in the lazy controls and the time-effect yielded another +22% increase in strength in those rodents who trained for 12 and not just 8 weeks.

Figure 2: Strength gains (left) and increases in cross sectional area, as well as intramuscular IGF1, myogenin and myoD expression (data adapted from Aguiar. 2013)

Now you may have heard all that before, what really makes this study stand out, however, is the observation of statistically highly significant correlations of intra-muscular IGF1, myogenin and myoD  mRNA expression, which speaks in favor of my previous hypothesis (read up on that in the Intermittent Thoughts on Building Muscle) that muscle growth is triggered, driven and maintained almost exclusively at a local level.

What are myogenin and myoD? Both are myogenic regulation factors with myogenin actually being part of the myoD family of transcription factors that will make stem cells develop into myocytes (myo D is highest in recently activated satellite cells).

So, when you are looking for "hormonal" (or other pro-anabolic) ghosts (Phillips. 2013), it is imperative to look for them right where the spook, or, in this case, the muscle building magic happens. If you do just that (see figure 2) and correlate the intra-muscular mRNA expression of IGF-1, myogenin and myoD, you will find the "ghostly" explanation for strength and size gains, as well as the confounding structural changes in the architecture of the muscle, with corresponding correlations between the increases in muscle cross-sectional area (CSA) of r = 0.85 (p = 0.0001), r = 0.87 (p = 0.0001) and r = 0.88 (p = 0.0001) for myoD, myogenin and IGF-1, respectively.

Fiber type changes take their time and occur only within the type II spectrum

A neat side-finding, which is actually no news, though, pertains to the fiber-type conversions that took place in response to the exercise regimen. Firstly, the scientists confirmed the notion that these changes occur exclusively within a certain fiber type. In other words, while Aguiar et al. observed conversions from the metabollically more flexible type IIX/D to the highly glycolytic (power) IIA type, no conversions of the highly oxidative type I to type II fibers were observed. And though the results would generally suggest that fiber IIX/D-to-IIA type conversion, as they 

"[...] also appear to occur during endurance training in humans, so that it would [be] reasonable to think that any exercise stimulus (e. g., endurance or strength) that is sufficient in duration and/or intensity can potentially induce conversions within the fast fiber population from type IIX/D to type IIA" (Aguiar. 2013),

the time-frame in the course of which these changes took place -- namely 12 weeks -- would confirm that the common fear of strength and endurance athletes could provoke negative structral adaptations from doing a "cardio" or "strength" workout from time actually is actually unwarranted. Neither will the former turn a powerlifter into a weakling, nor will the latter make a marathon runner "bulky". Both powerlifter and marathoner are on the contrary going to benefit from the conditioning effect and increase in strength, respectively -- not to mention the important effects on overall health both and not as mainstream stupidity will tell you only the powerlifter can derive from, figuratively speaking, "killing some game in the other's territory"

Bottom line: More food for intermittent thoughts on building muscle ;-)

Figure 3: Correlations between acute GH (A), free testosterone (B), IGF-1 (C)  and cortisol (D) responses (area under the curve—AUC) and gains in type II fibre CSA (Burd. 2013).

Eventually, this study is an excellent example of a way to design a rodent study in a way that will render its results actually meaningful. And what's more, in this particularly case these results are not just meaningful, but can also help us to make some sense of a couple of things we have not fully understood / appreciated, as of yet.

What I am particularly thinking about here, is the contrast between the in-vitro effects of IGF-1 and the (more or less absent) real-world effects of the IGF-1 response to exercise (=systemic increase), as it was observed by West and Phillips in a 2013 study. In their well-powered longitudinal study, neither the acute increase in systemic testosterone, nor the exercise induced increases in systemic IGF-1 showed significant correlations with the gains in type II CSA in a cohort (n = 56) of young men in response to 12 weeks of resistance training (West. 2013; see figure 3).

Another interesting finding of the West study was that, contrary to the circulating testosterone and IGF-1 levels, GH and cortisol did show direct correlations with increased muscle cross sectional areas in type II fibers.

And while the former correlation may be explained by the influence of growth hormone (GH) on the local expression of IGF-1 (Hameed . 2004), there is another open question left: How does cortisol actually figure in here? I mean, the chronic elevation / exogenous adminstration of cortisol, has been show to do the exact opposite, i.e. it decreases the local IGF-1 mRNA expression (Inder. 2010).

Figure 4: Graphical summary of what you should have learned Intermittent Thoughts on Building Muscle ... you didn't 'cause you are new to the SuppVersity or simply forgot about it? No problem read the preliminary summary and browse the individual chapters here!

What was missing in the Inder study, however, was the exercise component: Working out does not just exert protective effects against the negative side effects of the provision of exogenous "cortisol" (in this case Dexamethason), as they were observed in the afore referenced study by Inder et al., exercise will also lead to profound increases in local IGF-1 mRNA expression (e.g. +60% in Bamann. 2001), despite the fact that it will also increase the release of the falsely vilified anti-inflammatory glucocorticoid, cortisol... acute vs. chronic, local vs. system, peak values and amplitudes vs. plateaus and AUC values - you got to keep all these contrastive, yet complementary pairs in mind, when you are thinking about the endocrine and intracrine (within the cell) mediators of skeletal muscle hypertrophy.... what? Sounds familiar? Well, you must have been following the Intermittent Thoughts on Building Muscle, then ;-)

References:

• Aguiar AF, Vechetti-Júnior IJ, Alves de Souza RW, Castan EP, Milanezi-Aguiar RC, Padovani CR, Carvalho RF, Silva MD. Myogenin, MyoD and IGF-I Regulate Muscle Mass but not Fiber-type Conversion during Resistance Training in Rats. Int J Sports Med. 2013 Oct 11.
• 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.
• Ding H, Gao XL, Hirschberg R, Vadgama JV, Kopple JD. Impaired actions of insulin-like growth factor 1 on protein Synthesis and degradation in skeletal muscle of rats with chronic renal failure. Evidence for a postreceptor defect. J Clin Invest. 1996 Feb 15;97(4):1064-75. 
• Inder WJ, Jang C, Obeyesekere VR, Alford FP. Dexamethasone administration inhibits skeletal muscle expression of the androgen receptor and IGF-1--implications for steroid-induced myopathy. Clin Endocrinol (Oxf). 2010 Jul;73(1):126-32.
• Phillips SM. Strength and hypertrophy with resistance training: chasing a hormonal ghost. Eur J Appl Physiol. 2013 May;112(5):1981-3-
• Sculthorpe N, Solomon AM, Sinanan AC, Bouloux PM, Grace F, Lewis MP. Androgens affect myogenesis in vitro and increase local IGF-1 expression. Med Sci Sports Exerc. 2013 Apr;44(4):610-5.
• Tamaki T, Uchiyama S, Nakano S. A weight-lifting exercise model for inducing hypertrophy in the hindlimb muscles of rats. Med Sci Sports Exerc. 1992 Aug;24(8):881-6.
• 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. 2013 Jul;112(7):2693-702. 

80% Greater Protein Synthesis 3-5h After Workout: 20g+ PWO Protein Threshold Holds. Spiking Lower Amounts With Leucine or EAAs Will Still Yield Sub-Optimal Results

Image 1: Milk (proteins) are not just leucine or EAA - try doing that with half the amount of free-form aminos in water - the results will certainly be "suboptimal", I can vouch for that  ;-)

It has been a while since the last study from Stuart Phillips group at the McMaster University has made it to the SuppVersity news. Their latest publication does yet have the potential to pour oil on troubled waters, because the results appear to confirm that even when every other supplement appears to be failing you, you can always rely on your postworkout whey protein (Churchward-Venne. 2013). And while the study confirms that with some free form amino acid witchcraft, you can actually illicit identical post-exercise increases in protein synthesis, the previously determined threshold dosage of 20g of high quality protein is still the gold standard, for everyone whose interest is to actually build muscle which is, as you as a seasoned SuppVersity veteran know, not happening only in the first hour after a workout but within a 24h+  "window of opportunity" that has the size of barn door (cf. "Opening the 'Anabolic Barn Door' With the Key of Exercise and Nutrition Science!")!

You won't get a-whey without 20g+ of whey!

To elucidate whether the increasingly popular practice of pimping whole proteins with amino acids does make any sense in terms of being able to get away with less total protein, yet identical increases in post-workout protein synthesis Churchward-Venne et al. recruited 24 recreationally active, young adult male volunteers (22±0.6 years; 1.80±0.02m; 76.4±2.0 kg; BMI 24.3kg/m²), who had to perform a standardized 4x4 unilateral leg-workout with 3 min rest between sets consisting of

• 4x 10-12 reps of seated knee-extension and 
• 4x 10-12 reps of leg-press

at ~95% of their individual 10-rep max, which had been determined in a testing session 14 days prior.

Note: The reason Churchward-Venne et al. decided to use a unilateral exercise protocol was that this allowed them to take biopsies from both the exercised and non-exercised leg and thus determine the individual influence of exercise and supplementation.

You better make sure you get your protein, not just EAAs or leucine

Figure 1: Amino acid compositions of the test drinks (Churchward-Venne. 2013)

The study participants, who had consumed a standardized, prepackaged relatively low-protein diet (15% protein,. 55% carbohydrate, 30% fat) the day before the exercise intervention, were randomly assigned to consume one of the following drinks

• whey protein - 25 g whey protein isolate (total leucine: 3g)
• whey + leucine: 6.25 g whey protein isolate supplemented with free-form leucine (total leucine: 3g)
• whey + EAA: 6.25 g whey protein isolate supplemented with free-form EAAs (total leucine: 0.75g)

The 300ml of fluid which contained identical tracers, were consumed immediately post workout, blood and muscle biopsies were taken at regular intervals pre- and post workout and MPS, signaling through mTOR, and amino acid transporter (AAT) mRNA abundance were determined.

Figure 2: Relative expression of p-mTOR (left) and p70S6K (right) compared to baseline (Churchward-Venne. 2013)

Now what is interesting is that the "classic" markers of protein anabolism, p-akt (not shown, but exhibited significant differences between treatment), mTOR and P70S6K (figure 2 & 3) do not show a clear-cut advantage of either of the treatments. Immediately post exercise, the increase in mTOR in the exercised leg, for example, is significantly more pronounced in those subjects who consumed a whey protein shake. The "downstream" activation of p70S6K, which supposedly controls protein synthesis at the ribosome, however, is identical in all groups.

Figure 3: Pseudo (=simply weighed by the timespan) area under the curve (a.u) for mTOR and p70S6K, AUC for leucine (a.u.) and fractional protein synthesis in the exercised leg 3-5h after the workout (based on Churchward-Venne. 2013)

The same is true for the protein synthetic response measured as fractional protein synthesis in the whole post exercise period in the untrained, and up to 3h post exercise in the trained leg. Then, however, we see a markedly higher influx of protein into the trained muscle in the whey protein group, which is - and this is somewhat remarkable - not in accordance with the p70S6K levels, which would suggest that the protein influx should be maximal in the leucine and not in the whey group.

A protein pump without protein is useless

Based on the data we have, it is difficult to say whether it is the lack of an individual, a certain combination or the total amount of (non-)essential amino acids that is responsible for this affect. If you take a look at the amino acid composition of the test solutions in figure 1, it does yet appear likely to assume that it is the absence of non-essential amino acids...what? Glutamin? No, I thought so as well, but when you come to think about it, glutamine, of which we have recently seen that it does play a hitherto under-appreciated role in protein synthesis, is unlikely to exert this effect on its own. After all, Chiu et al. based their conclusions with respect to the necessity of glutamine to maximize protein synthesis on increases in mTOR expression (cf. "A New Role for Glutamine in Protein Synthesis?"). 3-5h after the workout the initially increased mTOR levels in the whey protein group had yet returned to baseline and the the leucine, BCAA and EAA levels in the blood of the subjects were identical in all groups (data not shown); and still, the influx of protein into the exercised leg musculature of the whey group was ~80% higher than that in the EAA group.

Image 2 (dormtainment.com): Subjects from the EAA, the leucine and the whey group (from left to right) after ingestion of the respective fluids - just kiddin' *rofl*

But let's be honest, in the end, these results only what common sense should have told us all along: You can push the gas pedal as much as you want (leucine group) and still won't get very far if your protein tank is half empty. Similarly, you can ingest as much leucine as you want and it will still have little effect on total protein synthesis, regardless of whether you train or not. For the practitioner, any further speculations about the minimal amount of leucine, a given persons in a given age-group would need to maximally stimulate protein synthesis, as the authors make them in their discussion of the results are non-significant compared to the following straight forward take home messages:

1. 20-25g of whey protein are still the go to post-workout protein source
2. building a better post workout protein from free form EAAs is not feasible
3. the importance of the non-essential amino acids in "real" protein is probably under-appreciated
4. the importance or I should say potency of leucine is probably much over-estimated
5. muscle protein synthesis and thus skeletal muscle hypertrophy is not a 2h post workout game

In essence, it would suffice to remember just (1) and (5) and to follow the simple yet effective maxime to get 20g+ of quality protein (not 20g leucine ;-) with every meal to get big and muscular and, as all of you who read yesterday's news or one of the many previous posts in which I envoked the findings of Loenneke et al. which show just that: People with a frequent intake of quality protein have the lowest body fat levels (Loenneke. 2013).

References:

1. Chiu M, Tardito S, Barilli A, Bianchi MG, Dall'asta V, Bussolati O. Glutamine stimulates mTORC1 independent of the cell content of essential amino acids. Amino Acids. 2013 May 8. [Epub ahead of print]
2. Churchward-Venne TA, Burd NA, Mitchell CJ, West DW, Philp A, Marcotte GR, Baker SK, Baar K, Phillips SM. Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men. J Physiol. 2013 Mar 25.
3. Loenneke JP, Wilson JM, Manninen AH, Wray ME, Barnes JT, Pujol TJ. Quality protein intake is inversely related with abdominal fat. Nutr Metab (Lond). 2013 Jan 27;9(1):5.
4. Moore DR, Robinson MJ, Fry JL, Tang JE, Glover EI, Wilkinson SB, Prior T, Tarnopolsky MA, Phillips SM. Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. Am J Clin Nutr. 2009 Jan;89(1):161-8.