Understanding Muscle Hypertrophy - Study Sheds More Light on Process of Satellite Cell Recruitement: SRF, IL-6, STAT3, COX2, IL4 + More Funky Acronyms With Important Roles in the Structural Component of Muscle Growth.

No pain inflammation, no gain? In the long(er) run this could in fact be true.

All of you who followed my advice to "like" the SuppVersity Facebook page and are thus keeping up with the numerous additional news I am posting there, should actually have seen the news item on the non-significance of the exercise-induced interleukin-6 (IL6) response for the exercise induced improvements in glucose metabolism (read more). The mere fact that the glucose metabolism of IL6(-) mice, which are mice who simply cannot express IL6, is still improved by "working out" does yet by no means preclude that the demonized cytokine does play a fundamental role in the exercise-induced systemic and local benefits. In fact, an even more recent rodent study would suggest that a certain degree of inflammation and the respective increase in IL6 immediately after a workout is even essential for persistent skeletal muscle hypertrophy.

As you may remember from the Intermittent Thoughts on Building Muscle Series there is more to skeletal muscle hypertrophy than the simple messages such as "increases protein synthesis by X%" that are printed in shiny letters on the boxes of hundreds of the currently available "natural muscle builders" on the real and digital shelves of the supplement vendors. One of these "mores" is the recruitement of satellite cells, muscle stem cells that are incorporated into the musculature to replace damaged myonuclei or increase the myonuclear density to allow for greater protein accretion (learn more).

Decreasing domain sizes = better function + higher growth propensity

"Hold on those are rodents and rodent studies are not relevant!" While it is a good thing to critically assess whether the results of a certain study can be species specific. The contemporary practice to question all rodent studies which are not part of your own cherry picked arsenal is getting onto my nerves. So, please check out the pretty analogues (short term unfortunately) human study by McCay from 2009 (McCay. 2009), before you stop reading after spotting the word "rat" in this article.

It is this process of satellite cell activation and incorporation of which Gwenaelle Begue and her colleagues from the University of Montpelier have now confirmed that it depends on the activation of the IL-6/STAT1/STAT3 signaling pathway in a prolonged 10 weeks resistance training scenario. In the course of the latter,  36 male Wistar rats were randomly assigned to one out of the following six groups:

• CTL2, CTL4, CTL10 (CTL = non-training controls, n = 6 in each group) and 
• TR2, TR4 and TR10, which were rats trained for 2, 4 and 10 weeks. 

The rodents in the TR-X groups were supposed to climb an apparatus with initially 50% later up to 210% of their body weight strapped to their back, five times a week. The load was increased every two days, if the rodents still managed to do "10 reps" = climb 10 steps and reached quite impressive levels of 120% of the body weight after two, 150% of the body weight after four and 210% after ten weeks of training.

Where is the rodent squat machine?

No rodent squat in the study at hand, but the "stair climbing" is a better full-body workout, anyways.

Now, this may not be as "realistic" a program as the rodent squat Aguiar et al. used in their 2013 study, but is is - and this is interesting - very similar to a test that has been done by many researchers with myostatin negative mice. As you will remember from the Intermittent Thoughts on Building Muscle Series (click here to read the pertinent part) those heavily muscled mice are unable to lift their own bodyweight, mainly because of the fact that the myonuclear domains within their muscle grew beyond a threshold where they absence of an adequate number of myonuclei per volume unit enders the muscle useless.

"Healthy" muscle growth does therefore require both, protein synthesis (increase in volume), as well as structural adaptations, so that the domain size does remain constant - at least!

"10 weeks of resistance training did not affect the myonuclear domain"

Against that background the last subheading, which is in fact a direct citation from the full text of the Begue paper is - contrary to what a non-SuppVersity reader could believe - good news. Very good news, to be precise:

Figure 1: Changes in fiber type ratios (left), cross sectional diameter according to fiber type (middle) and  fiber area per myonucleus (right; Begue. 2013)

As you can see in figure 1 (right hand side), there was even a small, yet statistically non-significant decrease in the fiber area each myonucleus had to control and that despite quite impressive increases of 77%, 92% and 100% in the cross-section of the type-I, type-IIa and type-IIx fibers of the animals (figure 1, middle).

Satellite cell recruitment, necessary of optional if you want to get big?

In this context, Begue et al. speficially point out that the "recruitment of additional nuclei derived from SC incorporated into muscle fibers" occurs parallel to the better known "resistance training induced enhancement of protein synthesis" that occurs "after the training session and last[s] up to 24–48 h in humans" (Bengue.2013). 

"Indeed, several works in humans have evidenced an increase in the number of myonuclei per fiber when fiber size increases approximately more than 25% (Kadi. 2004; Petrella. 2008). Thus, the myonuclear domain (i.e. the theoretical amount of cytoplasm supported by a single myonucleus in a muscle fiber) remained constant although a large increase in fiber CSA via the addition of SC-derived nuclei occurs." (Begue. 2013)

Since estrogen plays an important role in the regeneration of the satellite cell pool, it's pretty likely that you can literally "SERM your growth potential away" (learn more)

Notwithstanding the heavily quoted results of the 2011 study by McCarthy et al. in which the reasearchers were able to demonstrate that rodent muscle can grow even when it is satellite cell depleted, my personal conviction is that the latter process, i.e. the incorporation of new (not just even the replacement of damaged myonuclei is an obligatory prerequisite for persistent gains.

With +40% increased domain sizes, after only two weeks, it would have been interested to see how things would have developed in the subsequent weeks. I bet(!), the normal mice would have kept growing while their satellite cell depleted peers would have hit a plateau, where their own body woul have pulled the emergency brake aka myostatin (in this context, it's also interesting to remark that myostatin stops the proliferation of satellite cells and does thus indirectly divert the existing ones towards differentiation and incorporation into the muscle, cf. figure 2)

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Bottom line: The study at hand delivers further evidence for the intimate connection between "inflammation" or rather the expression of the still demonized inflammatory cytokine interleukin-6 and the incorporation of "fresh" satellite cells into the muscle. With the latter being a necessary prerequisite to keep the domain sizes within functionally optimal limits while the cross section of the fibers is expanding (the muscle is growing), it is likely an (I want to emphasis that!) not yet disproven that continuous muscle growth requires satellite cell recruitment.

Basically you can think of it like the Army. While it is (or at least has historically been) relatively easy to find any recruits (=increase protein synthesis), people who are qualified to become officers and coordinate the actions of the rank and file are hard to find and without an adequate number of them you will end up with a chaotic mess instead of a powerful army. That's actually pretty much what happens to the myostatin negative mice, who may be able to recruit officers,... ah, I mean to recruit satellite cells, but simply outgrow the maximal pace of satellite cell incorporation.

Figure 2: IL-6 is the first myokine you should remember, it "wakes" the quiescent satellite cells up, he COX-2 activated IL-4 is myokine #2 and initiates the differentiation / incorporation process which will eventually result in the formation of a new nucleus. .

What, oh yes, of course! I had almost forgotten the unfortunately quite complicated connection to IL-6. If you take a parting look at the figure on the right, you will realize that a diagram explains things much better than I could. In fact, the "motor" of the whole growth business is the contraction induced expression of serum responsive factor, of which Guerci et al. have found in 2013 that it is the previously missing link between muscular contractions on the one hand and the expression of myokines, who happen to be the same molecules we know as "inflammatory cytokines" in other contexts. Il-6 and the COX-2 activated IL-4 are then getting things rolling (Guerci. 2013)... what? No, I cannot tell you whether taking antioxidants will block that, but I can promise you that you will learn more about this tie-in within the next 7 days, so stay tuned ;-)

What I can tell you in advance, though, is that strength and size gains of IL-6(-) mice are compromised (Serrano. 2008). So even if I would have to qualify my previous statement that satellite cells are necessary for continuous growth - one thing is sure: Their activation by IL-6 is necessary for optimal growth.

References:

• Begue G, Douillard A, Galbes O, Rossano B, Vernus B, Candau R, Py G. Early Activation of Rat Skeletal Muscle IL-6/STAT1/STAT3 Dependent Gene Expression in Resistance Exercise Linked to Hypertrophy. PLoS One. 2013;8(2):e57141. 
• Guerci A, Lahoute C, Hébrard S, Collard L, Graindorge D, Favier M, Cagnard N, Batonnet-Pichon S, Précigout G, Garcia L, Tuil D, Daegelen D, Sotiropoulos A. Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy. Cell Metab. 2013 Jan 4;15(1):25-37.
• Kadi F, Schjerling P, Andersen LL, Charifi N, Madsen JL. The effects of heavy resistance training and detraining on satellite cells in human skeletal muscles. J Physiol. 2004; 558: 1005–1012.
• McCarthy JJ, Mula J, Miyazaki M, Erfani R, Garrison K. Effective fiber hypertrophy in satellite cell-depleted skeletal muscle. Development. 2011; 138: 3657–3666
• 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. doi: 10.1371/journal.pone.0006027.
• Petrella JK, Kim JS, Mayhew DL, Cross JM, Bamman MM. Potent myofiber hypertrophy during resistance training in humans is associated with satellite cell-mediated myonuclear addition: a cluster analysis. J Appl Physiol. 2008. 104: 1736–1742
• Serrano AL, Baeza-Raja B, Perdiguero E, Jardí M, Muñoz-Cánoves P. Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy. Cell Metab. 2008 Jan;7(1):33-44.

Easy Whey to Prevent LPS Induced Inflammation? Whey Protein Prevents LPS Binding to TRL-4 and IL-8 Production. Surprise: Pressurized Denatured Whey Works Best!

Unlikely that this or whatever whey protein you have bought as of late was pressurized with an Avure High Pressure Processing System at more than 500mPa in order to denature it (yeah you're reading right) and produce a bunch of fancy new peptides which appear to have even more potent anti-LPS effects than those in regular whey protein (hydrolysate).

Saturday's post on saturated fatty acids and their negative effects on post-prandial endotoxemia has turned out to be (un-)surprisingly popular. I am still not sure if this will be same for the post at hand; and that despite the fact that it revolves around the exact same topic, namely the inflammatory reaction to lipopolysaccharides (LPS). In the case of endotoxins such as LPS, the inflammation, which is, as you all should by now be aware of, an endogenous "alert, defense and repair" reaction of our bodies, is actually triggered by their interaction with the so-called toll-like-receptor. Now scientists from the McGill University in Montreal have found that there is a substance all of you are familiar with and some of you may even be consuming on daily or at least regular basis that can block this interaction, as well as the ensuing overproduction of cytokines and/or other well-meant, but in the end potentially hazardous immune responses.

There is al-wheys something new about whey ;-)

As the researchers point out the beneficial effects of whey proteins don't just go way beyond their muscle building effects and are mediated by several and not just one of it's amino acid, protein and peptide ingredients:

"Whey proteins (WP), a by-product of the cheese-making industry, possess nutritional benefits as a source of protein of high biological value. Whey products and whey-derived peptides have demonstrated a number of anti-inflammatory effects. These anti-inflammatory effects include decreased cytokine release in rodent models of ischaemia– reperfusion and exposure to LPS. In addition, individual whey constituents, such as lactoferrin or glycomacropeptide, and peptides released from these by pepsin– pancreatin hydrolysis exhibit anti-inflammatory effects, such as suppression of tissue neutrophilia or inhibition of inflammatory cytokine release." (Iskandar. 2013)

In that, whey proteins have been shown to be particularly useful for the treatment and/or management of chronic inflammatory diseases such as cystic fibrosis, a disease passed down through families that causes thick, sticky mucus to build up in the lungs, digestive tract, and other areas of the body and a disease that will probably make you - just like me - think about the high cysteine content of whey, immediately.

"Under pressure..."

For the study at hand the researchers used two different epithelial cell types and added lipopsaccharides (LPS), as well as

• regular whey (Inpro 90 Whey Protein Isolate from Vitalus Nutrition) that had been enzymatically hydrolysated (=predigested) to yield a product that would be similar to what many supplement are now selling you as either intra- or post-workout "super whey" (their not my claim ;-), or
• pressurized whey, which was based on the same raw material, but was pressurized before being hydrolysated

to their petri dishes and observed the effect the additional whey proteins had on the LPS-induced interleukin-8 (IL-8) production and the binding of the Escherichia coli LPS to the TRL4s (toll-like receptor 4) on the surface of the epithelial cells.

Figure 1: Differential effects of different doses (in µg/ml) regular and pressurized whey protein hydrolysate on LPS-induced IL-8 secretion in 1HAEo- cells (left) and corresponding LPS-binding to toll-like receptors on the surface of the cells (right); data expressed relative to LPS only (Iskandar. 2013)

As the data in figure 1 clearly shows, both whey protein hydrolysate, the regular, as well as the pressurized one had similar effects on the binding of LPS. The ensuing decrease in cytokine production (IL-8) was yet statistically significant only in the dish with the pressurized whey protein hydrolysate. Moreover at the highest dosage of the normal whey protein hydrolysate, there is what you could call a "rebound effect", if this reached statistical significance - which it obviously didn't.

Figure 2: Effect of 500 or 1000mg/ml of pressurized whey protein and native whey protein hydrolysates on 1HAEo- cell culture medium ferric-reducing antioxidant power  (FRAP); data expressed relative to basal levels (Iskandar. 2013)

That said, the overall effect size dependent not just on the type of whey protein hydrolysate, but also on the cell type: While the 1HAEOo- cells (shown in figure 1) needed the highest tested dose to show statistically significant reductions in IL-8 production, the effect reached significance at 500 µg/ml for the CFTE29o- cells using the pressurized whey protein hydrolysate and at 1,000µg/ml for the regular WPH (obviously no rebound here).

A similar difference was observed in the results FRAP essay (FRAB stands for ferric reducing ability of plasma and the results provide information about the general antioxidant defenses of the cells). While the CFTE29o- cells (shown in figure 2) were happy with both whey protein hydrolysate, the pressurized WPH had a minimal, but statistically non-significant edge in the 1HAEOo- cells.

Is there anything special about pressurized hydrolysates?

In previous studies the researchers had already established that pressurization of WP improves its
in vitro digestibility, promotes the release of novel peptides by gastrointestinal digestive enzymes and enhances the antiinflammatory effect (Vilela. 2006).

"These in vitro findings were also confirmed in clinical studies. Thus, a 2-week supplementation with pressurised whey increased the levels of glutathione, a crucial low-molecular anantioxidant, in peripheral blood mononuclear cel. Further, we have reported that a 1-month dietary supplementation with pressurised whey improved nutritional status and markers of systemic inflammation in patients with CF [cystic fibrosis]." (Iskandar. 2013)

In the study at hand, the research team from Canada did now want to (a) investigate the potential anti-inflammatory and antioxidant effects of pressurized and regular whey protein hydrolysates in the context of cystic fibrosis and non- CF respiratory epithelial cells and (b) explore the mechanisms by which pressurised and native whey exert their beneficial anti-inflammatory effects. Their research hypothesis was that it is the difference in peptide (=complex bond of amino acids that has different effects from the same amino acids in isolation) availability that is enhanced by the pressurisation of whey that's responsible for it's superiority compared to regular whey proteins.

Establishing the (leaky) gut, bacteria, non-alcoholic fatty liver disease connection

With the researchers focus being on cystic fibrosis, the results of this in-vitro trial are still highly meaningful for all of us. In particular the more potent increase in overall anti-oxidant capacity upon exposure of the epithelial cell lines would suggest that the ingestion of pressurized whey proteins could exert similar benficial effects in other parts of the body, specifically the digestive tract, as well.

Suggested read: "Plus: 20+ Things to Protect and Restore the Integrity of Your Intestinal Wall" (read more)

On the other hand, the more important blockage of the toll-like receptors, was virtually identical with both the regular and the pressurized whey protein and the general implications of these findings are actually pretty far-reaching. After all, TRL4 has only recently been implicated in the development of fibrosis in non-alcoholic fatty liver disease subsequent to alteration of gut microbiota, increased intestinal permeability and the ensuing increase in exposure of the liver to gut-derived bacterial products (Frainarius. 2013). The exact same horror-scenario many of you will probably have had on mind, when they read about the effects the high saturated fat content had in the study by Mani et al. from Saturday.

Bottom line: So will just having your daily whey protect your gut from all assaults? Probably not, but is may be just another one of the many small things which may not render your intestinal wall and overall immune system bullet-proof but will at least help them to come with the omnipresent and 24/7 assault they are exposed to. Whether it's really got to be pressurized whey, on the other hand, remains questionable. Personally, I don't think so - if you take a look at the dose-dependently reduced expression of IL-8 in response to LPS exposure upon co-administration with pressurized and normal whey, it seems as if the pressurized variety did not only have the edge, but was also lacking the rebound effect that occurred at very high doses in the 1HAEo-cells.

If you want to live out your OCD tendencies on your whey intake, start with rule #1 "Never Sip Your Whey" (read why).

Since, previous have shown that pressurization does impart significant changes not just to the digestibility of whey protein hydrolysates, but also to their peptide structure (Vilela. 2006), it could thus well be that the observed differences are of real world importance, as well, and cannot be compensated for by just taking more of the "regular" whey protein hydrolysates.  I hope there will soon be comparative studies in human subjects available, until then I would not worry too much about not getting the "optimal" whey protein to combat TRL-4 binding of LPS and increase anti-oxidant defenses. As a regular SuppVersity reader, you are probably apart of the privileged part of the Western society which has to care least about LPS and overall (bad) inflammation, anyways. Plus, as the study goes to show "regular" whey has similar, albeit not "optimal" effects in this regard.

References:

• Iskandar MM, Dauletbaev N, Kubow S, Mawji N, Lands LC. Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4. Br J Nutr. 2013 Jan 3:1-11.
• Frasinariu OE, Ceccarelli S, Alisi A, Moraru E, Nobili V. Gut-liver axis and fibrosis in nonalcoholic fatty liver disease: An input for novel therapies. Dig Liver Dis. 2013 Dec 29.
• Vilela RM, Lands LC, Chan HM, Azadi B, Kubow S. High hydrostatic pressure enhances whey protein digestibility to generate whey peptides that improve glutathione status in CFTR-deficient lung epithelial cells. Mol Nutr Food Res. 2006 Nov;50(11):1013-29.