Intermittent Thoughts on Building Muscle: The Skeletal Muscle Hypertrophy 101 - Part 2: Getting Big Means Growing Beyond Temporary Physiological Limits.

Image 1: This is another type of "dysfunctional muscle"; distinct from the one we are talking about, here

Although I assume that you all have read the last installment of the Intermittent Thoughts, I took FatFree's comment that he (or she?) was missing the "too comlicated check box" from the old design to the heart (I do so with every comment, btw, so keep them coming) and kick off today's installment of "The Thoughts" with a brief and even more "dumbed down" summary of what we have learned about the (possibly) ascertained and, even if human skeletal muscle hyperplasia existed, dominant factors in the trinity of skeletal muscle growth: Protein synthestic increases in myonuclear domain sizes and the satellite cell driven incorporation of new myonuclei.

Getting big goes beyond ballooning up

You probably will remember the balloon-metaphor, I introduced in the red infobox toward the end of the last installment. Let's briefly get back to that and use it to reavaluate the results from the Quaisar study (Qaisar. 2011, see also "What is Hypertrophy"). Rizwan Qaisar and his colleagues from the Uppsala University in Sweden had analyzed the differential effect of insulin-like growth factor 1 (IGF1) over- and myostatin-underexpression on muscular size and function (the latter is important, since we know that a complete lack of the "muscle growth blocker" myostatin leads to huge, but disfunctional / weak muscles). 

Figure 1: Domain sizes of EDL and soleus muscle fibers in wild-type control, myostatin negative and IGF1 overexpressing mice (data based on Qaisar. 2011)

Now to really understand the meaning of what may be the main message of the study, it is imperative to understand the basic architecture of muscle fibers. If you think about a complete muscle fiber as a bundle of ballons that is wrapped into a strechable net, then each balloon would represent one myonuclear domain. The owe their name to the fact that they actually are the "domains" which surrounding a single myonucleus (lat. plural "myonuclei") within a given skeletal myocyte, which - contrary to other cells in your body - has the ability to hold multiple nuclei. Now, the most obvious determinant of the myonuclear domain size is the ratio of protein in- to protein efflux. And it is this connection on which researchers base their belief that by simply measuring the protein synthetic response to exercise and/or supplementation would suffice to predict long-term increases in muscle size (and subsequently strength / performance).

Growth is limited and myostatin is not just a pain in the ass of anyone who wants to "get big"

Image 2: Sketch of a mammalian skeletal muscle fiber - myonucleus (turqouis), mitochondria (blue),  sarcoplasmic rectilium (buff), tubules (orange), myofibrils (pinkish)  - Artist: Lesley Skeates. Originally from Gray's Anatomy 29th ed. Elsevier. 2008

If you take another look at the data from the Qaisar study (cf. figure 1), you will notice that uncontrolled growth in "one dimension", i.e. exclusive increases in domain size, generates larger muscles, but at the same time renders them dysfunctional, a process of which Qaisar and his colleagues believe that it is caused by a decrease in the number of strongly attached cross-bridges, which are the primary source of the small specific force in muscle fibers with very large MNDs. This hypothesis is by the way supported by a lowered myosin (contractive motor-protein) content per muscle volume in the myostatin negative mice.

In order to really understand why this is the case, it may help if you take a look at the (awesome) sketch of a mammalian skeletal muscle fiber on the right (image 2). The myonuclei are colored in turquois-green, they are connected to the mitochondrion (blue) and the sarcoplasmic rectilium (buff), which is traversed by transverse tubules (orange, and not easy to distinguish). The major part of the muscle fiber is yet made up by myofibrils, which are protein chains containing actin, myosin, and titin...

Ah... wtf. Before someone wants to click the "too complicated"-button again, let's just say the myofibrils are the ones who do the actual work. Now, with the increased domain size (obviously the domain is the "zone" comprising all the aforementioned components that a single nucleus is "responsible" for) and the consequent decrease in myosin content (per volume), as well as the reduced number / density of cross-bridges, i.e. links between the myofibrils to coordinate their action, the muscle loses its functionality. Just like a labor brigade with 5 smaller, smart guys who listen precicely to what their foreman says and work hand in hand can get the job done more efficiently than 5 big, but dump guys, who do not even listen to what their foreman tells them, this type of one-dimensional growth, i.e. an exclusive increase in domain sizes, goes at the expense of muscle function.

From satellite cells to broadcasting towers and back again

Image 3: Myonuclei have a domain, similar to the broadcasting area of a transmitter mast.

So, the myonuclear domain, has nothing to do with some sort of "fenced off" area that is protected by a cell membrane. In physics, we would probably talk about a field, a field of influence, just like an electromagnetic field, with the exception that the signalling from the nucleus does not work via EM radiation, but via gene-signalling... mTOR & Co says hello ;-) Satellite cell recruitment and the "installation" of new myonuclei would thusly be equal to the installation of new broadcasting towers, which make the existing system more effective and allow for further expansion. If you are a mobile communications veteran, who knows the "good" (or rather bad) old days of poor reception you'll know what I mean.

In this context it may be interesting that a very recent study by Antonios Matsakas et al. was able to show that the voluntary wheel running or swimming was able to restore the function of the "over-blown" muscle of myostatin-null (MSTN-) mice (Matsakas. 2011). Another clear cut evidence that exercise induces structural changes which go well beyond the accrual of protein that is not adequately controlled in the MSTN- mice.

Do you take my point now? Ok, then let's get on...

The expression of myostatin, which prevents the myonuclear domains from further expansion is thusly a means by which your body maintains muscular function. Contrary to my friend Adelfo, your body has no interest in looking like Phil Heath... the only reason it has to grow is to be able to survive and survival requires functional strength, not size. There is yet some leeway as far as increase in domain sizes are concerned and it is this leeway that explains the "exorbitant" gains you have been making when you first hit the gym. The lazy bastard (sorry ;-) you have been before, your myonuclear domains were probably far beyond their "functional" limit and, consequent to the acute protein synthetic response to your 1001 biceps curls, "ballooned up" until, just as Darryn S. Willoughby observed it in his 2004 study (Willoughby. 2004), the contemporary increase in skeletal muscle myostatin content brought the expansion of the "bloated" myonuclear domains to an "abrupt halt" (at least that was probably your perception).

Figure 2: Relative myofibrillar protein content and myostatin mRNA expression in the thigh muscles of 11 previously untrained subjects in response to a 12-week (3x per week) resistance training regimen with 3 sets of leg presses and knee extensions à 6-8 reps @85-95% of the 1RM (data calculated based on Willoughby. 2004)

As the relative amount of myofibrillar protein content and myostatin mRNA expression in skeletal muscle from the previously 22 untrained male subjects of the Willoughby study shows (cf. figure 2), the myofibrillar protein accretion is accompanied by profound increases in the expression of muscular myostatin. Or, put simply, the bodies of the subjects, whose thigh volume increase by roughly 16% in the course of the 12-week study period, were sensing that without structural changes, this type of muscle growth would eventually lead to huge, yet dysfunctional muscle fibers - something that obviously would not promote survival and is thusly not part of our genetic program.

Structural changes facilitate new growth

On the other hand, the constant overload to which (I hope) you are exposing yourself in the gym signals your body that without increasing strength (again, your body does not care about size), it will not last much longer is the "adverse environment" of the gym. So, the only way to "survive" is to rebuild / restructure the muscles, a process of which we have seen in the last installment of this series that it goes hand in hand with decreases in the number of purported hypertrophy prone "ultra-fast" twitch type IIb muscle fibers (or rather the content of respective myosin heavy chains within your muscles). Whether the resulting phenotype is that of a bodybuilder, characterized by increases in both the number and size of slow-twitch type I and fast-twitch (intermediate) type II-x fibers, or that of a powerlifter, characterized mainly by increases in the number and size of (intermediate) type II-x fibers, depends on the training stimulus, alone:

Image 4: Our bodies respond to different training routines by distinct changes in the muscular structure.

• Wanna get strong like a German Olympic gold medalist Matthias Steiner? Then goto the gym, 10x a day and do a 1-rep max plus minimal auxiliary work like people the Bulgarian O-lifters are supposed to do. 
• Wanna get big like Arnold? Then follow his example and break into your local gym on Sunday (Arnold's was not open 24/7 back in the day, but that did not stop him from training) and pump out rep after rep, after rep to make sure your body understands that you want to maximize both type-I as well as type-II fiber size.

This does not mean that you cannot get big and strong, it does yet mean that a competitive bodybuilder will - per pound of lean body mass - always be weaker than a powerlifter.

"I need YOU!" ... to pick my brain and steer this series in the right direction

The sixty-four-thousand-dollar question now is: What is the "best" way to let your body know what you (not even your brain, but rather your mind) wants? And even at the risk that I am losing my "guru status" now, I want to be honest with you: I don't know the answer... at least not yet ;-) I thusly depend on your help, on people like Steven Acerra, who is constantly picking my brain with interesting questions and studies on facebook, Mike T Nelson, who lately jumped in on an interesting discussion on training stimuli, "Fat Free", Aaron, Matt, Erik Istre, Lerner (whose comments I have been missing lately) and all the rest of you who chime in with questions, suggestions or the simple assessment that the "good Dr. Andro" is once again making things only more complicated ;-)

And as a food for thought, I give you a sneak peak at what should come next in this series: It is the intricate relation of protein and endocrine signaling by which your muscles and no central governor or transient elevations in isolated systemic testosterone, growth hormone or insulin levels regulate the concomitant increases in muscle protein synthesis and satellite cell recruitement and changes in the myosin heavy chain composition. So, assuming that this installment of the Intermittent Thoughts did not raise further questions as far as the basics are concerned, the next installment will revolve around the role of IGF1 and its local (=intra-muscular) cousins MGF and IGF-IEa, which appear to play a key role in the the coordination of the restructuring process that will keep your muscles functional, even when you are approaching a Olympia stage ready bodybuilding physique.

Intermittent Thoughts on Building Muscle: The Skeletal Muscle Hypertrophy 101 - Part 1: What is Hypertrophy?

Image 1: Governator, Arnold Schwarzenegger, as a teen and in his early twenties. What - puberty aside - were the underlying mechanisms of the obvious increase in sleeve-size? I mean on a strictly myocellular level, of course ;-)

Those of you, who have been following the Intermittent Thoughts series over the last weeks will probably already have realized that I finally dropped the "Intermittent Fasting" from the title... this is something I have been thinking about for quite some time now. After all, the series has evolved way beyond its initial focus on a specific dietary protocol and has become more of an educational "how do you find your way to success"-series. With the format, intermittent thoughts, being still the same, the upcoming posts in this series, which will certainly touch on the topic of intermittent fasting, again, will be titled "Intermittent Thoughts on..." whatever the topic of the week may be... and for this week's installment the latter is going to be "Building Muscle: The Skeletal Muscle Hypertrophy 101".

I can already foresee that this is only the first in yet another series of posts. An introduction into the myocellular mechanisms that turn a normal teen like into a symbol of physical culture, or, to put it simply:  

What actually is skeletal muscle hypertrophy? 

Before I even try to answer this question let me remind you of something you have learned about "growth" in one of the previous installments of this series. In "Building Muscle Starts With Losing Weight" you have learned that one of the greatest fallacies of "classical" bulking, as in "eating everything that cannot escape your ravenous hunger for mass", is adipocyte hyperplasia. You may also remember that this increase in the number of fat cells occurs, when your existing fat stores are ready to burst and your body is in need of new storage capacities. Analogously, you would expect your muscle fibers to "hypertrophy" (from mechanical overload and constant nutrient abundance) until they are "ready to burst" and then divide and form new muscle fibers. (Un?)fortunately, myocytes are not adipocytes and thusly things are working somewhat different, here.

Figure 1: Overview over the three (?) pathways by which your skeletal muscles "grow".

If you take a look at the graphical overview I have come up with in figure 1, you will see that there are two, maybe three distinct pathways which contribute to what the average trainee subsumes under "hypertrophy".

1. Pathway A - hypertrophy via satellite cell recruitment and increases in the number of myonuclei per muscle fiber,
2. Pathway B - hypertrophy via increases in myonuclear domain size within an existing muscle fiber, and
3. Pathway C - hyperplasia, which would be the increase in muscle size by cell division and thusly an increase in the number of muscle fibers

The existence of different fiber types, or to be precise, the co-existence of different fiber types (fast twitch, slow twitch and various sub-types) at varying ratios within a single muscle group complicate things even further. Instead of giving you the usual theoretical lowdown on type I and type II fibers and how the former are supposedly used for endurance and the latter for strength training, I want to discuss the matter from a more practical perspective and first pose the question: What is that we actually want? 

I mean, you do not want "hypertrophy", but you want to get big and buffed, right?

Assuming that this is the case we should initially define "big and buffed" on a myofibrillar level by taking a look at how the muscles of the forerunners of physical culture actually look like - and I promise, what you will be learning today will, once again(!), go against conventional wisdom. Or would you have expected that bodybuilding is a sport that is characterized by a loss in highly glycolytic type IIb fibers and increases in both the intermediate type IIa, as well as the "endurance type" slow-twitch muscles? No? Well, then you should have a look at the data in figure 2:

Figure 2: Fiber composition of bodybuilders, recreational lifters, endurance rowers and sedentary control; determined via myosin heavy chain (MHC) isoform content of the triceps brachii muscle (data adapted from Jurimäe. 1997)

The results of the 1997 muscle biopsies by Jurimäe et al.  (cf. figure 2) clearly show that "getting big and jacked" is by no means about maximizing the "hypertrophy-prone type II fibers", as you may have read it numerous times on one of the thousand bulletin boards, or the numerous blogs of self-proclaimed fitness experts (Jurimäe. 1997). A bodybuilder is rather a person who has maximized the expression of myosin heavy chain I and IIa. Specifically with reference to the latter, the authors write:

It is interesting to note that Kraemer et al. (1995) have reported a lack of change in the area of fibres consisting predominantly of MHC type IIb proteins (i.e. FTb fibres) as a consequence of a 12-week resistance training programme. This suggests that a shift from MHC type IIb proteins to type IIa MHC isoforms may be a necessary prerequisite for FT fibre hypertrophy to occur. Consistent with this was the significant negative correlation (r = -0.67) between the percentage of MHC type IIb isoforms and arm circumference. Similarly, the smaller arm girth of the C group may have been partially due to the greater content of MHC type IIb isoforms in this group.

Or put simply,  the "strong" type IIb fibers have a very limited (if any) propensity for hypertrophy. So that, in order to maximize growth, it is necessary to trigger a shift towards the more "intermediate" type IIa fibers. If you take into consideration, how almost all bodybuilders got, where they are now, i.e. by a volume training approach, this is actually something you should have been able to infer simply from what has been and is still working for 99% of the trainees.

Figure 3: Intercorrelations between myosin heavy chain (MHC) isoforms and isoinertial (1-RM max), isometric (extension) and isokinetic (extension peak torque) strength indices (data adapted from Jurimäe. 1997)

Moreover, the data in figure 3 shows that this does not necessarily mean that they have to sacrifice their strength, as the percentage of type IIa fibers does not only correlate with increased muscle size, but also with increased isoinertial (1-RM max, r=0.66), isometric (workload, r=0.51) and isokinetic (peak torque, 0.61). strength. Getting big and buffed and getting strong thusly both require a profound shift in the "God given" fiber composition, but why?

Skeletal muscle hyperplasia - yes or  no? While there are a handful of studies which speak of hyperplastic responses to stretch or other form artificial overload, many (if not all) of them have been done on avian myofibers (Kelly. 1996), which, due to their special make-up, make it a) very difficult to distinguish between increasing overlap due to the longitudinal growth of intrafascicularly terminating skeletal muscle fibers and "real" hyperplasia and b) may not even translate to human beings. That's the reason, why I will disregard the issue of hyperplasia in the following discussion.

A very recent study by an international group of scientists from Sweden and the USA, may provide some insights, into why these fiber-transformations are necessary if you want to grow tree-trunk legs and sleeve-bursting arms. In this study, which was published on November 28, 2011, in the FASEB Journal (Qaisar. 2011), Rizwan Qaisar and his colleagues provide a detailed analysis of the the muscle fiber composition of mice who are either myostatin-negative or over-express the muscle building growth hormone IGF1 (we are talking about intra-muscular IGF1, here! More on that in future installments of the series).

Figure 4: Cross sectional area (CSA), number of mynuclei and myonuclear domain size of myostatin negative mice and mice overexpressing IGF1 relative to wild-type control (data calculated based on Qaisar. 2011)

As you would expect, both the myostatin-negative, as well as the IGF1 mice were more muscular than their wild-type cousins. There were, as you can see in figure 4, yet significant differences in fiber sizes (CSA), the number of myonuclei per fiber, and the domain sizes of the individual myonuclei in the exclusively fast-twitch extensor digitorum longus (EDL) and the predominantly slow-twitch soleus muscle.

Image 2: The balloon metaphor of skeletal muscle hypertrophy.

Note: If you picture a muscle fiber as a number of balloons which are held together by an elastic net, then the myonuclei would be within the individual balloons, which, in turn, would represent the myonuclear domains. You could thusly increase the muscle size, i.e. stretch the net, by either inflating the balloons, i.e. increasing the domain size, or simply adding more balloons to the net. The latter would then be equivalent to the recruitement of new myonuclei from the satellite cell pool in the sarcoplasma of the muscle fibers.

If you take a closer look at the data you will notice that in the IGF1 mice the predominantly fast-twitch EDL muscle growths mainly by increases in myonuclei number, a feature that is absent in the predominantly slow-twitch soleus fibers. The profound increases in myonuclear domain size that occur in the myostatin-negative mice, on the other hand, result in profound reductions in muscle function.

Figure 5: Specific force, stiffness and myosin content (secondary axes) of EDL and soleus muscle in wild-type control, myostatin negative and IGF1 overexpressing mice (data based on Qaisar. 2011)

Both specific force, as well as as muscle stiffness, are profoundly reduced in the EDL muscle of the myostatin negative mice (cf. figure 5), because they have surpassed the maximally sustainable domain size and have thusly become dysfunctional.

Figure 6: Domain sizes of EDL and soleus muscle fibers in wild-type control, myostatin negative and IGF1 overexpressing mice (data based on Qaisar. 2011)

This becomes even more obvious if we take a look at the domain sizes in isolation (cf. figure 6). It is the "uncontrolled" growth that is partly a result of a lack of satellite cell recruitment and consequent increases in myonuclei number, which cripples most of the animals with mutations in the myostatin gene.

Muscle hypertrophy = increases in myonuclear number & domain size

Healthy muscle growth, that is the intermittent take-away of this installment of the Intermittent Thoughts on building muscle, is thusly a direct result of "hypertrophy", as it is commonly associated with increased protein synthesis (and decreased or constant protein breakdown) and the subsequent expansions of individual myonuclear domains and the recruitement of satellite cells, which will then form new myonuclei.

As you may have noticed from the increasing amount of typos, of which I have probably overlooked 50% (sorry for that), my Sunday time-budget is already exhausted, so that I will have to postpone the discussion of what triggers these processes to the next installment. I do yet hope that the stuff you learned today provides enough food for thought to get you through the week ;-)

HIT Your Satellite Cells to Increase Your Gains! Only High Intensity "Cardio" Exercise Will Fuel Your Satellite Cell Pool and Set You Up For Future Muscle Growth.

Image 1: NO-mediated satellite cell
recruitement (Anderson. 2000)

You have read it on the SuppVersity, you have heard about it on Carl Lanore's Super Human Radio and the BodyRX Show and those of you who have seen videos or pictures from the latest New York City Marathon, should actually have been able to infer it from the way the "finishers" looked like. Intensity not duration is what counts, when doing "cardio". Yet, as a very recent (7 days old) study shows (Naito. 2011), High Intensity Training (HIT) will not only burn off your lovehandles, while keeping your muscles intact, it will also prime your musclefibers for future growth by increasing the number of satellite cells, the small dormant mononuclear progenitor cells that are sandwiched between the basement membrane and sarcolemma of the fibers of your muscle and are recruited, whenever your body feels that you could use a little more or have to replace some damaged muscle mass.

While I will go into more detail on how your muscles actually grow in the upcoming parts of the Intermittent Fasting Series, in the course of which I am going to explain how you should train, eat and sleep in order to exploit all three major pathways of skeletal muscle growth, I want to give you a sneak peak at what you are going to learn, by highlighting that protein synthesis, i.e. the accrual of muscle protein in existing myonuclear domains, and the recruitment of satellite cells to replace damaged or add new myonuclei are distinct processes. It should nevertheless be obvious that with all the protein synthesis of the world you will - sooner or later - hit a plateau, when all the existing myonuclei have "blown up" to their maximal size - or as Naito et al. put it: "Increases in the number of satellite cells are necessary for full skeletal muscle growth and hypertrophy" So, whenever the existing myonuclei have reached their "full potential", the only way to keep growing is by adding new myonuclei via satellite cell recruitment. Keep that in mind before you discard the results the following study, because the "HIT rats" did not gain more "active" muscle than the "LIT rats" ;-)

In their experiment Hasashi Naito and his colleagues from the Tokai University and the Juntendo University in Japan put 17-week old (these are old rats!) female Sprague-Dawley rats on one out of four exercise regimen (for a detailed outline of the regimen, cf. table 1):

1. High Intensity, High Duration (90H)
2. High Intensity, Low Duration (30H)
3. Low Intensity, High Duration (90L)
4. Low Intensity, Low Duration (30L)

Table 1: Outline of the exercise
protocol (from Naito. 2011)

In the course of the 10-week study period the rats were exercised five times a week on one of those funky rodent treadmills. What's funny is that despite the fact that, as the scientists say, "[e]lectrical shocks were used sparingly to motivate the animals to run", two of the critters in the high intensity groups refused to do their workouts, which reminds me of what Dr. Layne Norton had to say on one of the past installments of BodyRX Radio: "Most of those who will tell you that they cannot do HIT for whatever reasons are usually just too lazy" - we may thus consider those two lazy rats as evidence for the accuracy of the model... and by the way, it did not save them from being anesthetized and deprived of their plantaris muscle, which was weighed and analyzed for its fiber composition and satellite cell count.

As it was to be expected in view of the high age of the rats, where skeletal muscle mass maintenance, may be considered a success, there were no statistically significant increases in plantaris and/or body mass in any of the treatment groups.

Figure 1: Changes (compared to untrained control) in number of myonuclei and satellite cells per muscle fiber (data calculate base on Naito. 2011)

Despite the absence of measurable skeletal muscle hypertrophy, the pronounced (cf. figure 1) and fiber-type specific (cf. figure 2) increases in satellite cell counts in the high intensity groups may well be considered as the necessary prestage of a hypertophic growth spurt, which could be triggered by appropriate training (which would obviously be strength training) and endocrine (more on that in the conclusion) stimuli.

Figure 1: Satellite cells per muscle fiber in type I (slow twitch) and type II (fast twitch) muscle fibers of rats in the control and the high intensity, high duration (90H) groups (data calculate base on Naito. 2011)

In that, it is also interesting to note that contrary to popular believe, the slow-twitch type I fibers, with their greater number of satellite cells, have an increased propensity for maximal myonuclear numbers, the fable of the "hypertrophy-prone fast-twitch type II" fibers, on the other end, is a consequence of their ability to accumulate more protein per myonucleus. And while I will - as promised in the red box above - dig deeper into that in future installments of Sunday's Intermittent Thoughts, I can already tell you that the fiber composition (not the size!) of professional body builders is almost identical to those of non-strength-trained individuals and thusly fundamentally different from that of strength athletes, like powerlifters (Tesch. 1982) - in order to achieve maximal muscularity you can thusly not neglect your type I fibers!

That being said, both the strength training, which would make use of the increased propensity to grow by recruiting satellite cells to form new myonuclei, as well as the necessary local IGF and MGF responses, which have been shown to decrease with age (Grounds. 2002), were absent in the study at hand. In someone like you, a young, vigorous strength trainee, both stimuli will yet obviously be present in abundance (at least I would hope so ;-). Accordingly, 1-3 high intensity (and in view of the fact that the duration, 30 vs. 90min, did not make a difference probably also high intensity interval) training (HIT or HIIT) sessions per week could not only make your increasingly fat-free muscles shine in their full glory, they will also "precondition" you for future muscle growth by increasing your satellite cell pool. I would thus suggest, you better not join the two lazy rats from the study, and rather find yourself the next best track to do a bunch of sprints ;-)

Low Testosterone ⇨ No Muscle Repair & Long-Term Gains: Testosterone's Effects on Satellite Cells & Myonuclei. Plus: Paradoxical 19% Drop in T W/ Whey + CHO Shake in Boys

Studies show that "women demonstrate significantly larger satellite cell and satellite cell nucleus areas than men" (Roth. 2000) - an effect that could be mediated by the effects of estrogen, not testosterone on satellite cells.

As a SuppVersity reader you will know that satellite cells are the "muscle reserve" that's "cooking" between the basal lamina and sarcolemma of your muscles. They are the cells your body needs to (a) repair damaged muscle cell nuclei (myonuclei) and (b) generate new myonuclei. As a SuppVersity reader you will also know that these new myonuclei are necessary to "Grow Beyond Temporary Physiological Limits" (learn more) that are imposed to your muscle by the myostatin increase that occurs, when the domain size, i.e. the volume of your muscle that's controlled by a single myonucleus, becomes too large.

To make a long story short: Without satellite cells, your muscle repair and growth will be impaired, which is why the results Thue Kvorning and Danish colleagues present in a soon-to-be-published paper in Acta Physiologica are relevant for everyone who want to gain or simply maintain maximal / optimal muscle mass.

You can learn more about satellice cells & co at the SuppVersity

Acc. Satellite Cell Growth

Training in Hypoxia

Epicatechin as a Muscle Builder

Intracrine Effects of Anabolics

NAC Impairs Sa- tellite Cell Act.

Understanding Muscle Growth

In the corresponding experiment, the Danish researchers treated 22 moderately trained young men with a GnHR analogue called goserelin that will dock to the gonadotropin receptors in the brain without stimulating the production of testosterone.

Figure 1: Graphical overview of the procedures (Kvorning. 2014)

Needless to say that the testosterone levels of the study participants were significantly reduced after 4 weeks, when the actual resistance training study began - see Figure 1 for an overview.

In obese boys whey + cho shakes lead to sign. T-reductions (Schwartz. 2014)

Speaking of ways to reduce testosterone: A recent study in obese pubertal boys showed - much to my own astonishment - that having 30 whey isolate + 30g glucose will significantly reduce the testosterone levels (19%) of the young subjects.

Don't ask me what exactly it is that causes this effect, but I suspect it may be related to the huge insulin spike (+410% - no typo!) the subjects experienced. From studies in women with polycystic ovary syndrome we know that insulin which will usually augment the release of sex hormones loses its function in vivo (Willis. 1996).

A similar temporary suppression of the pulsatile luteinizing hormone release and subsequent acute redutions in testosterone has been observed in adults in response to glucose alone (Iranmanesh. 2012). No reason to avoid carbs, though. In a study by Volek et al. the consumption of a high fat diet lead to a persistent 22% and 23% reduction in total and free testosterone (Volek. 2001).

A standardized training program which didn't look much different from what some of you may be doing at the gym (both the guys in the active and the placebo group had to perform, by the way):

"A standardized warm-up was performed before training consisting of 4 sets of squats with 20 repetitions without load and with 1 min rest between sets. Subjects from both groups trained using the same progressive strength training program. The programs were performed 3 times a week for 8 weeks and consisted of leg press, knee extension, leg curl, bench press, lat pull down, biceps curl, and elbow extension. Subjects did 4 sets of each exercise for the lower body and 3 sets of each exercise for the upper body. The strength training period consisted of 24 training sessions periodized in 3 cycles of 8 training sessions with changing training loads (6 RM – 10 RM). The goserelin group and the placebo group increased training loads to the same extent and underwent the same training volume." (Kvorning. 2014)

In contrast to the training stimulus, which was identical for both groups, the intra-muscular response to the workout differed significantly between the young men in the goserelin group (10-20x reduced testosterone levels) and their peers in the placebo group:

Figure 2: This is one of the cases, where having more is not better. Having more free satellite cells and fewer myonuclei is certainly not a good thing for someone striving to build maximal muscle mass.

In spite of the fact that in both, the placebo and goserelin groups, training lead to a signficant increase in the number of satellite cells in fast twitch type II fibers by 20 % in placebo and by 52 % in goserelin (p<0.01), the number of myonuclei, which is the one that's important with respect to future growth remained unchanged in the goserelin (p<0.05) group. In the placebo group, on the other hand, the myonuclear number increased significantly by 12 %.

In view of the resistance training focus of the workouts, it's not really surprising that on such changes were observed in the slow-twitch, endurance-type type I fibers in either group.

Can't believe estrogen is required for muscle building, check out the previous evidence in this SuppVersity article!

Bottom line: In view of preceding evidence that it's not testosterone, but estrogen that's required for proper satellite cell function (see "Estrogen, Friend or Foe of Skeletal Muscle Hypertrophy? Plus: Hey, Bro! Are You 'SERMing' Away Your Satellite Cells?" | learn more), it's a pity Kvorning et al. didn't measure the estrogen levels as well.

In view of the fact that men produce their estrogen via aromatization from testosterone, it's yet very likely that not just the T, but also the E2 levels of the men in the goserelin group were significantly suppressed. The association between estrogen and bone health and the distinctive changes in bone morphogenetic proteins signaling the researchers observed would actually support this hypothesis, which should remind you of the fallacy of abusing SERMs and / or aromatase inhibitors longer than it would be necessary to normalize your estrogen levels, guys.

And I mean, if you have enough testosterone, you'll have enough estrogen, as well, right? Comment on Facebook!

PS: I am fully aware that the necessity of satellite cell recruitment for muscle gains (and by some scientists even repair) is still debated (cf. Pallafacchina. 2013), but up to now, I still have to see the counter-evidence that is not based on mere short-term muscle protein synthesis studies. If you look at the mechanism, it should be obvious that satellite cell activity becomes important only, when the natural upper limit for functional domain sizes is reached and that was certainly not the case in any of the commonly cited rodent studies. Plus: There is evidence in favor of the important role of satellite cells in skeletal muscle hypertrophy from other studies (Appell. 1988; Schultz. 1989; Rosenblatt. 1994;  Barton-Davis. 1999; Mitchell. 2001).

References:

• Appell, H-J., S. Forsberg, and W. Hollmann. "Satellite cell activation in human skeletal muscle after training: evidence for muscle fiber neoformation." International journal of sports medicine 9.04 (1988): 297-299.
• Barton-Davis, E. R., D. I. Shoturma, and H. L. Sweeney. "Contribution of satellite cells to IGF-I induced hypertrophy of skeletal muscle." Acta physiologica scandinavica 167.4 (1999): 301-305. 
• Iranmanesh, Ali, Donna Lawson, and Johannes D. Veldhuis. "Glucose ingestion acutely lowers pulsatile LH and basal testosterone secretion in men." American Journal of Physiology-Endocrinology and Metabolism 302.6 (2012): E724-E730.
• Kvorning, Thue, et al. "The activity of satellite cells and myonuclei following 8 weeks of strength training in young men with suppressed testosterone levels." Acta Physiologica (2014). 
• Mitchell, Patrick O., and Grace K. Pavlath. "A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy." American Journal of Physiology-Cell Physiology 281.5 (2001): C1706-C1715. 
• Pallafacchina, G., B. Blaauw, and S. Schiaffino. "Role of satellite cells in muscle growth and maintenance of muscle mass." Nutrition, Metabolism and Cardiovascular Diseases 23 (2013): S12-S18.
• Rosenblatt, J. David, David Yong, and David J. Parry. "Satellite cell activity is required for hypertrophy of overloaded adult rat muscle." Muscle & nerve 17.6 (1994): 608-613. 
• Schultz, E. D. W. A. R. D. "Satellite cell behavior during skeletal muscle growth and regeneration." Medicine and science in sports and exercise 21.5 Suppl (1989): S181-6.
• Schwartz et al. "Acute decrease in serum testosterone after a mixed glucose and protein beverage in obese peripubertal boys." Clinical Endocrinology (2014). Accepted Article. 
• Volek, Jeff S., et al. "Effects of a high-fat diet on postabsorptive and postprandial testosterone responses to a fat-rich meal." Metabolism 50.11 (2001): 1351-1355.
• Willis, D. E. B. B. I. E., et al. "Modulation by insulin of follicle-stimulating hormone and luteinizing hormone actions in human granulosa cells of normal and polycystic ovaries." The Journal of Clinical Endocrinology & Metabolism 81.1 (1996): 302-309.

Anabolic Workouts Revisited - Testosterone, GH, Prolactin & Co: Differential Effects of Workout Type, Volume & Density

Ronnie Coleman is just one of the pros who trained with crazily high volume and weights - is that the way to go, or does it require too many "supplements"?

"What's the most anabolic form of training?" To answer that question we would actually have to initially define how we are planning to measure "anabolism". If we go by the more or less confuted paradigm that the immediate hormonal response to a workout is one, if not the, fundamental determinant of it's effectiveness, today's blogpost provides you with a whole host of already known and novel insights that could come handy, when you're setting up your next workout routine.

Please keep in mind, though, that the study by West et al., in which the researchers found no correlation between exercise induced increases in testosterone and only weak correlations between growth hormone and fast twitch muscle fiber size and cortisol and overall lean mass, over a 15-week period, as well as my dissertations on the complexity of "building muscle" in the Intermittent Thoughts on Building Muscle, while I am taking you through the latest results.

Part I - Shorter rest times = greater "anabolism"

I'd like to start out with the results,Villaneuva et al. recently published in the Journal of Strength and Conditioning Research (Villanueva. 2012). The scientists from the University of Southern California conducted a 2x2 randomized trial involving strength and hypertrophy oriented total body workouts on Tuff Stuff Performance Series equipment with different different set/rep schemes and rest times, but identical exercises for both:

maximum strength protocol (S) 8 x 3 (sets x reps) at 85% of predetermined 1-RM for all resistance training exercises; 60 seconds (S60) or 90 seconds (S90) rest in-between sets muscular hypertrophy protocol (H) 3 x 10 (sets x reps) at 70% 1-RM for all resistance training exercises; : performed with either 60 seconds (H60) or 90 seconds (H90) rest in-between sets

Exercises smith machine barbell back squat flat barbell bench press  narrow/neutral grip lat pulldown  seated unilateral knee extension

The six study participants were young men, who volunteered for the study (age 26+/-2.4 years, 178.6 +/-5.9 cm, and 86.4+/-1.2kg) were healthy and described as "recreational resistance trainees who trained at least 2 days per week (>2 years)" (Villanueva. 2012).None of them was yet a competitive weight lifters or did engaged in any other sport-specific training. Furthermore none of the 6 participants took any medication or dietary supplements that could potentially have skewed the results, i.e. the change in cortisol and total testosterone levels, I plotted in figure 1:

Figure 1: Change of total serum testosterone concentration from rest (PRE) to immediately post-exercise (POST), Pre to 15 minutes post-exercise (15 MIN), and Pre to 30 minutes post-exercise (30 MIN) for strength and hypertrophy protocols with short (60s) and "long" (90s) rest period (* indicates significant difference - p < 0.05; based on Viallaneuva. 2012).

The overall message here should be clear: If you want to ramp up your testosterone levels, hit it fast and in the classic hypertrophy range with 3 sets of 10 reps!

Exercise-induced increase in testosterone = anabolism?

While we cannot exclude that the exercise induced increase in testosterone (and other "anabolic" hormones) is a necessary and facilitative part of the adaptive cascade at the end of which you may in fact have gained another inch on your arm, the results of the initially mentioned study from Phillips lab at the McMaster University in Ontario, do at least suggest that there is no linear, nor otherwise proportional relationship between exercise induced increases in testosterone and gains in lean muscle mass (West. 2012).

Figure 2: Differential endocrine response to high vs. low volume squatting in healthy trained men - androgen receptor content of vastus lateralis (left), total testosterone levels (right; data based on Rattames. 2005)

The notion that intracrine (=within the cells) processes, proteins and hormones are the actual driving forces and controlling factors in skeletal muscle growth is further supported by observations Ratamess et al. made in 2005. The researchers from the Human Performance Laboratory at the University of Connecticut found a 45% reduction in androgen receptor expression in response to high (figure 2, MS: 6x10, 2min rest in-between) vs. low volume (figure 2, SS: 1x10) squatting sessions in healthy, resistance-trained men with a minimum 3 years of experience with the back squat exercise. At first sight, this does certainly seem as if the testosterone response was pretty useless, after all, even this proven muscle builder (see "Zoning In On the Big T" and "Quantifying The Big T") cannot do its muscle building job, if there is no receptor to bind to, right?  Correct! ... but follow up studies by Spiering et al. and Vingren et al. have shown that the testosterone release in and out of itself will illicit increases in receptor density which do however need some time to take place: In that, the +14% increase in testosterone in response to a high volume upper body workout in the Spiering study increased the androgen receptor expression 3h after the workout by 40% over control (Spiering. 2009).

Strength vs. Hypertrophy - Check... Endurance exercise & Sprinting?

Before we discuss the implications of these findings, let's briefly take a look at another even more recent study trying to discover the complicated hormonal response to different types of exercise that could help us to grasp a better notion of "The latest on working out for anabolism". Now, I would venture the guess that 99% of you are probably thinking about "3,2,1" vs. "5x5" vs. "HST" and a couple of other classic strength training or bodybuilding routines, right now. The results the already mentioned study which have been published only 4 days ago in the Journal of Applied Physiology do yet bring two unexpected training types to the play: Sprinting and endurance training. I see, you are surprised. Well, at least for the first one, i.e. sprinting, you actually should not be; after all, I have been writing about the protein anabolic effects of HIIT (=multiple sprints) before (see "The Anabolic Effects of HIIT"). So, if it increases muscle protein synthesis by >40%, why shouldn't sprinting also be able to establish an overall more anabolic milieu? But endurance training?

Figure 3: Prolactin, insulin (in the absence of a "control" AUC, I calculated the value relative to the mean), testosterone, cortisol and growth hormone (GH) area under the curve (pre to 60min post) in 8 healthy young men in response to 4 separate trials involving resting (control, relative to which all the other values - except insulin - are expressed to), resistance training, sprinting and endurance training (for details see text; data based on Stokes. 2012)

Yeah, it sound counterintuitive, but if you take a close enough look at the data in figure 3 and take into account growth hormone (GH) bars at the right hand side of the colored graph have their own scale you will have to concede that the 8.4x increase in growth hormone expression in response to 30min cycling at 70% of the VO2max are pretty impressive. Specifically, if you consider that the competition, i.e. a...

*the scientists write it the other way around, but honestly I have not et come across a study, where the rest between sets was longer than the one between exercises, so I assume this is a typo

• 30min total-body resistance training regimen - bench press, leg press, bench pull; 75% 1RM 5 sets 10 reps, each; 60s rest* between sets, 180s rest* between exercises, and an
• all out 30s sprint that was performed subsequent to a warm-up that consisted of cycling for 4 min at 60 W, 30 s at 80 W, and
  then 30 s at 100 W and a 5-min pause on a friction-loaded cycle ergometer at 7.5 % (75 N/kN) of the subject’s body mass

were training regimen you would probably rather associate with the term "growth" as in "growth hormone". Now, if you ask the "bros" about their reasons for taking GH, I guess that few of them will still be falling for the idea that it was a great muscle builder (when used appropriately, it can be a exponentiate the anabolic effects of superphysiological doses of testosterone, though).

Most of them will probably tell you that it helps you lean out... and why does it do that? Simply because it helps with fatty acid mobilization and oxidation. And when is the need for the latter the greatest? When you sleep, yeah... when you fast, ok... but also when you expend huge amounts of energy from fat! And that's exactly what's happening during the 30min of cycling at 70% of the VO2 max, an undertaking that does not simply burn relatively, but most importantly absolutely significantly more energy and thus fat, than either the strength training (82% less energy expenditure) session, or the sprint session (94% less energy expenditure).

Fine, so a drop in FFA will increase GH, but what's that about prolactin?

Figure 4: GH response of young men in response to exhaustive endurance (15min, targer HR 160bpm) and resistance training (total-body, 5 exercises, 4 sets classic pyramid, 1-2min rest) and combined training (crossfit-style + 6x10m sprints) from Akbari. 2012

These remarks on growth hormone do yet not answer a question of which I suppose that it's already preying on your minds: What's that about sprinting induced increases in prolactin? Well, if

• higher volume weight lifting in the hypertrophy range (8-12 reps) increases testosterone, and
• energy consuming endurance exercises with a tendency to reduce free fatty acids in the purported "fat burning zone" (70% VO2Max) increases GH

the straight forward answer to this question would be: A ...

• higher mean work rate [=energy expenditure per time unit of exercise] maintained only over a very short timespan increases prolactin

And just to make that clear, the work rate during the sprint was 430% higher than during the resistance training session and still 260% greater than during the 30min of cycling.

On a side note: The drop in insulin after the endurance trial (>60% immediately post) supports the view that the associated GH response is primarily fat-catabolic and not muscle anabolic. Moreover, it's IGF-1 it's splice variants MGF & Co, not it's parent GH which are mainly responsible for the muscle building effect. And at least the synthesis of IGF-1 is in a hitherto not fully elucidated way related to insulin (click here to learn more), the villain of the last decade that was once, and is still hailed among many hardcore bodybuilders as "the most anabolic hormone of all"!

In view of the fact that this rise in prolactin (actually somewhat of an acute stress response, of which we don't yet know what exactly it's role wrt to training adaptation is) went hand in hand with a temporary increase in insulin this may sound as if sprinting was a bad thing, but the spike in insulin may have been significant compared to the steady insulin levels of the control, let alone the declining insulin levels in the endurance trials, but was almost identical to the one that occurred in response to the resistance training regimen (the AUC for insulin was even higher in the 60min after the resistance training protocol). And has, as Stokes et al. point out unquestionable benefits:

"Increased insulin concentrations as seen following the sprint trial [...] might facilitate muscle glycogen synthesis during recovery through insulin’s actions on both glucose transport and on glycogen synthase activity. The significant increase in insulin concentrations following sprint exercise in the present study is followed by a suppression of blood glucose concentrations to levels below pre-exercise. This finding might have implications for individuals who have difficulty regulating blood glucose concentrations, such as individuals with impaired glucose tolerance." (Stokes. 2012; my emphases)

The post-exercise increase in the universally and wrongfully demonized insulin could thus not just come handy for the the 8 young recreationally active men who participated in the study at hand, but also for the average and extraordinary gymrat (like yourself?) and even the obese type II diabetic who has finally found his/her way to physical culture! And this is not simply a vague assumption, but an already empirically validated hypothesis (e.g. Richards. 2010; Whyte. 2010).

So what does all this tell us then?

Did we even answer our question? By now you should actually notice something. The question "What's the most anabolic workout?" cannot be answered with only one definition of "anabolism" on your mind. The classic body part split with 3-5 sets of 5-10 reps per exercise is probably still the way to go, if your perspective on anabolism relates to increased skeletal muscle hypertrophy based on both, endocrine (testosterone), as well as intracrine (mTOR) responses to your workouts. If you want to build your brain and thus interpret "anabolism" as "neurogenesis", you will have to either sprint or do extra-long endurance work (Rojas Vega. 2012) or have lot's of sex to up your prolactin levels . And if your mitochondrial density is what's on your mind, when you think of "anabolism", HIT and HIIT should be your best friends.

Aside from the time-delayed increase in testosterone receptor expression, of which you could either argue that it could be evidence for a shift from an intracrine to an endocrine anabolic response, and the beneficial effects of an increase in insulin at the right time, namely post-workout when your muscles are ready to take up the glucose that could otherwise end up being converted to triglycerides and stored in one of the numerous fat depots of your body, there are two other imho important reasons not to simply fall into the opposite extreme and say "Wtf if exercise induced increases in anabolic hormones don't correlate with muscle growth they are completely worthless!":

1. Real world, not lab evidence: High(er) volume training with short(er) rest periods was and still is the way the majority of using and non-using athletes are "building" those physiques on, people actually have on their minds, when they are looking for the "most anabolic workout". 
2. Effects on intracrine factors of anabolism: From mTOR-dependent local protein synthetic response over the many, hitherto not fully understood intracrine growth factors, the role and function of inflammatory cytokines and the immune response to exercise, up to the maintenance and incorporation of satellite cells into new myonuclei and the PGC1-alpha driven increase in mitochondrial density, all of the things, we are training for show some correspondence with the exercise induced systemic expression of "anabolic" or "catabolic" hormones.

The main problem is therefore, as Stokes et al. state, that we still have an "over-simplistic" concept of the "anabolic (e.g. testosterone and growth hormone) and catabolic (e.g. cortisol) hormones" (Stokes. 2012), in which things like the prolactin response to exercise, which has only recently been implicated as a driving force of exercise induced neurogenesis (Rojas Vega. 2012), have not even had a place, until now... apropos prolactin, you should be aware that having an orgasm will likewise elicit a temporary spike in prolactin levels and sex is therefore "brain-o-bolic" (see my post on the matter on the SuppVersity Facebook wall)!

References:

• Akbari A, Mojtahedi H, Marandi SM, Movahedi A, Ramsheh SFR. Comparing the Effects of Three Types of Exercise (Exhaustive Endurance, Intensive Resistance and Combined Exercise) on the Secretion of Growth Hormone in Active Men. World Journal of Sport Sciences. 2012; 6 (3): 247-253. 
• Ratamess NA, Kraemer WJ, Volek JS, Maresh CM, Vanheest JL, Sharman MJ, Rubin MR, French DN, Vescovi JD, Silvestre R, Hatfield DL, Fleck SJ, Deschenes MR. Androgen receptor content following heavy resistance exercise in men. J Steroid Biochem Mol Biol. 2005 Jan;93(1):35-42.
• Richards JC, Johnson TK, Kuzma JN, Lonac MC, Schweder MM, Voyles WF, Bell C. Short-term sprint interval training increases insulin sensitivity in healthy adults but does not affect the thermogenic response to beta-adrenergic stimulation. J Phys-iol (Lond). 2010; 588(15):2961–2972 
• Rojas Vega S, Hollmann W, Struder HK. Influences of exercise and training on the circulating concentration of prolactin in humans. J Neuroendocrino. 2012; 24(3):395–402.
• 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.
• Stokes KA, Gilbert KL, Hall GM, Andrews RC, Thompson D. Different responses of selected hormones to three types of exercise in young men. Eur J Appl Physiol. 2012 Sep 13.
• Villanueva MG, Villanueva MG, Lane CJ, Schroeder ET. Influence of Rest Interval Length on Acute Testosterone and Cortisol Responses to Volume-Load Equated Total Body Hypertrophic and Strength Protocols. J Strength Cond Res. 2012 Jul 12. 
• 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. 
• Whyte LJ, Gill JMR, Cathcart AJ. Effect of 2 weeks of sprint interval training on health-related outcomes in sedentary over-weight/obese men. Metabol Clin Exp. 2010; 59(10):1421–1428.