Leucine, Citrulline or a Non-Essential Amino Acid Mix - Which Amino Acid(s) are Most Effective in Preventing Muscle Loss During an 18h (Intermittent) Fast?

Image 1: If Chris, "the Techician", Aceto's usually well-informed sources are right and the former Mr Olympia Jay Cutler is currently trying to lose muscle (I heard him say that on Heavy Muscle Radio), Cutler would be ill advised if he ingested ~20g of non-essential amino acids during and / or in-between extended fasts and hours of arduous low-intensity cardio sessions (img  MuscleTech)

Those of you who followed the "Amino Acids for Super Humans" series I did earlier this year on Carl Lanore's Super Human Radio may remember the arginine < > citrulline < > ornitine cycle and how I tried to explain that, from a physiological perspective, arginine's role in ammonia detox is probably as, if not more important than its role in the production of nitric oxide. What most of you will probably have overheard, or, in the respective shownotes, over-read, was my reference to a 2006 study from the University of Paris, which was - at least to my knowledge - the first study to show that citrulline (much like leucine) increases protein synthesis and thusly reduces the loss of muscle protein in old malnourished rats (Osowska. 2006). As it is often the case with isolated study results like that, these observations have not gotten much attention within the research community, so that it is not very surprising that the latest information on citrulline's putative role in whole body protein homeostasis come from the same laboratory at the Sorbonne, as the previously cited ones.

Citrulline vs. Leucine, and non-essential aminos as a control!?

What is particularly interesting about these results, the scientists from the Département Biologie Expérimentale, Métabolique et Clinique at the Pharmaceutical Faculty of the venerable Université Paris Descartes published in the (btw. highly recommendable) Journal Amino Acids, is that they allow for a direct comparison of the magnitude and the mechanism the ingestion of citrulline, leucine or a mix of other non-essential amino acids has on the fractional protein synthesis in skeletal muscle tissue (Tibialis anterior) in a fasted state (18h food deprivation).

Figure 1: Fractional protein synthesis (in %/h) in tibialis anterior muscle of fasted rats 50 minutes after administration of leucine, l-citrulline or isonitrogenous (to leucine) non-essential amino acids (data adapted from Plenier. 2011)

To my own surprise the winner of the battle of the "protein anabolic amino acids" is neither the usual (leucine), nor the unusual suspect (citrulline), but rather the non-essential amino acid combo which consisted of 1.35g/kg of alanine, glycine, proline, histidine, asparagine and serine.

Alanine, glycine, proline, histidine, asparagine, serine - Non-essential high potentials?

Let's briefly put this surprising result into (a human) perspective: If we assume that you are on an extended intermittent fast, traveling or had - for whatever other reason - no access to food for 18h, then the ingestion of 0.22g/kg of a non-essential amino acid mixture (if you weigh 80kg that would be 17.5g), would induce a 9.37% greater increase in muscle protein synthesis than the same amount of leucine and a 16.67% greater increase than 23g of l-citrulline.

Figure 1: Phosphorylation of Akt, s6K, 4EBP1 (left) and AMPK (right) 60min after administration of leucine, l-citrulline or isonitrogenous (to leucine) non-essential amino acids (data adapted from Plenier. 2011)

If we combine the previous calculations with the data from the Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component, it becomes even more obvious that this study provides further evidence against the current over-emphasis of l-leucine which is so prevalaent especially among the bodybuilding-oriented physical culturists. As I have pointed out in previous posts, here at the SuppVersity, pushing the "protein-anabolic gas-pedal" through the floor (=ingesting huge amounts of leucine on its own) makes no sense if your car has long run out of fuel (=there are no amino acids to synthesize).

Against that background it is actually not very surprising that the protein synthesis in the fasted leucine group was reduced, although the phosphorylation of  p70S6K was identical and the one of 4EBP1 even greater (both indicate that the protein synthetic machinery was set into gear) than in the fed control. What is surprising, though, is the fact that the actual protein synthetic response in the leucine group fell 10% short of the one that was observed in the tibialis muscle of the rodents which receive an isonutrogenous amount of non-essential amino acids. After all, previous studies have suggested that the induction of measurable increases in protein synthesis was an exclusive property only branched chain (BCAA) or essential (EAA) amino acid mixtures would posses. Methodological differences in the design of respective studies aside, Servane Lé Plenier and his colleagues suggest the following two possible explanations for the surprising effects the alanine, glycine, proline, histidin, asparagine and serine combo exhibited on skeletal muscle protein synthesis in the fasted state:

[firstly,] in the fasted state, NEAA homeostasis is maintained by catabolism of essential amino acids (EAA) - alanine, for example, is produced in muscle from LEU and pyruvate - and limited EAA availability affects MPS since it is well known that a deficiency in one amino acids may be a limiting step for protein synthesis. Hence, in the fasted state, NEAA administration could spare EAA utilization and thereby preserve MPS.

[secondly,] one or more amino acids in the NEAA mixture could display specific anabolic properties. For example, alanine has been shown to stimulate liver protein synthesis in starved rats (Perez-Sala. 1987), but to the best of our knowledge this effect has not been shown in muscle. Similarly, proline and glycine may possess pharmacological properties that could indirectly modulate protein synthesis.

Personally, I don't believe that any of the non-essential amino acids (NE-AA) in the NE-AA formula actually had an individual effect on protein synthesis beyond its ability to spare essential amino acids and its availability as a substrate for inter-organ amino acid transfer (especially for alanine and asparagine, which are transaminated in the liver, this could be an important factor). So that the practical implications of this study should be clear: if you want to minimize muscle loss during a(n) (intermittent) fast, you better have some non-essential amino acids with your leucine!

One question answered, 999 new ones raised

Image 2: If you have read all Intermittent Thoughts articles which dealt with the AMPK/mTOR Metabolic Seesaw and the respective follow-ups, you will probably already have noticed that the ingestion of non-essential amino acids had the least impact on the fasting-induced increase in AMPK-phosphorylation of all three treatments. And I guess I don't have to tell you that this is good news for all intermittent fasters out there - spare the muscle, improve your health and burn the fat, what more can you as for?

Unfortunately, this study leaves us with way more questions than answers. I personally, for example would venture the guess that the ingestion of a complete EAA product would result in an even more profound amelioration of the fasting induced reduction in fractional protein synthesis. That being said, the latter could also compromise another advantage of the non-essential amino acids, I have not even mentioned, yet: their almost non-existent effect on intra-muscular AMPK-expression (cf. figure 2, right). If you read all Intermittent Thoughts articles which dealt with the AMPK/mTOR Metabolic Seesaw and the respective follow-ups, you will be familiar with notion that the fasting-induced phosphorylation of intra-muscular AMPK is responsible for the majority of the health, as well as the closely related fat-burning effects of (intermittent) fasting. Now, if the ingestion of a ~20g bolus of alanine, glycine, proline, histidine, asparagine and serine could increase your skeletal muscle protein synthesis back to almost normal levels (NE-AA -12.5% vs. leucine-only -20%), while keeping the AMPK-alpha levels maxed out (cf. figure 2, right), it would at least warrant an experiment before we totally discard the possibility that, under certain circumstances, such as the fasting window of an intermittent fast, the oftentimes disregarded "non-essential amino acids" could perhaps be more than just a band-aid when you have run out of essential ones.

Whether there will be a place for citrulline in particular is questionable, though. With the least effect on protein synthesis and the greatest impact on AMPK, it would de facto be a "band-aid" solution, for everyone who fasts, deliberately. In other contexts, however, l-citrulline supplementation could well have its merits. In cancer patients it could for example be used to ameliorate muscle loss without triggering the pro-carcinogenic (Garcia-Maceira. 2009), but I guess this would be the topic of another study and another blogpost, here at the SuppVersity ;-)

Branched Chain Amino Magic: Study Takes Another Step Towards a Better Understanding of the Anabolic & Anticatabolic Effects of BCAAs and Their Essential Cousins

Image 1: Without the other essential amino acids (EAAs), the branched chain amino acids, leucine, isoleucine and valine (BCAAs) have nothing to "build" your muscle from ;-)

Usually, I do not get very excited, when I hit upon another study into the "protein-synthetic response" that is triggered by the ingestion of branched chain amino acids (BCAAs). I mean, let's be honest... we all know that their ingestion will trigger the phosphorylation of the mammalian target of rapamycin and thusly increase protein synthesis, so why would we need another study where instead of a 17.5% increase in protein synthesis, we would see a 18.3% increase? Actually, we don't... the data Marcus Borgenvik, William Apró and Eva Blomstrand from the  Åstrand Laboratory, Swedish School of Sport and Health Sciences and the Karolinska Institutet, in Stockholm, Sweden (Borgenvik. 2011), collected goes yet well beyond what we have seen in most of the previous studies and is thus well worth an individual blogpost here at the SuppVersity.

BCCAs work! How? Little do we know...

If we are honest, we must concede that our (=the scientific) understanding of the complex processes that are triggered when "large" amounts of BCAAs hit our bloodstream, is very limited. What we know is that we can measure increases in mTOR-expression that correlate with likewise measurable increases in protein synthesis. What we do not really know is how exactly one leads to the other and where the influences of amino acid supplementation and exercise training overlap. This is even more true for the complementary side of the protein synthetic equation of which Borgenvik et al. state that

[w]hereas extensive evidence for the stimulatory effect of amino acids, either alone or in combination with exercise, on protein synthesis has been reported, their effect on protein breakdown is elusive.

In that, it is particularly confusing that "previous investigations involving ingestion of essential amino acids (EAA) in connection with resistance exercise have revealed no attenuating effect on protein breakdown", while studies which investigated the effect of BCAA or leucine in isolation, report reduction in protein degradation in subjects at rest or performing eccentric endurance exercise (MacLean. 1994). Reason enough for the Swedish scientists to recruit a group of seven healthy, recreationally active participants (5 men, 2 women; 27 (± 2) years; height 175 (± 5) cm; weight 67 (± 7) kg), put them on a standardized diet (17% protein; 25% fat; 57% carbs; ~2100kcal for women, ~2700kcal for men) for two days and, on the subsequent morning (subjects reported to the lab fasted) and after a thorough warm-up, have them perform

• 4 sets of 10 repetitions at 80% of their predetermined 1 RM, followed by another 
• 4 sets of 15 repetitions at 65% of their 1 RM of single-legged leg presses.

The subjects used the same leg on all exercises and rested ~5min after each set. Before the warm-up, during and immediately after and 15 and 45min after the exercise regimen the subjects consumed either

• 150 mL of BCAAs (2:1:1 ratio) in flavored water, or
• 150 mL flavored water alone.

The total amount of BCAAs was 85mg/kg or 5.695g for the "average" study participant. After four weeks the experiment was repeated with each participant receiving the opposite treatment.

Figure 1: Complete analysis of serum amino acid levels in the trained and untrained leg of subjects receiving BCAA or Placebo supplement before, during and after the completion of a standardized single-legged leg press exercise (data adapted from Borgenvik. 2011)

As far as the study protocol goes this is thus certainly not an extraordinary study. If you take a look at figure 1, where I deliberately plotted all the data the scientists gathered as far as serum amino acid concentrations are concerned, you will yet realize that what makes this study stand out is the sheer amount of parameters Borgenik et al. have analyzed. Similar data is also available for the amino acid concentrations in the exercised muscle and though, the scientists, who set out to investigate the effects of BCAA supplementation on protein breakdown, would probably disagree with me, here, I feel that this data actually has the most real world significance for physical culturists, like you and me.

Figure 2: Relative increase / decrease in intra-muscular BCAAs and other EAAs in BCAA supplemented subjects vs. placebo control at different time-points before, during and after single-legged leg presses (data adapted from Borgenvik. 2011)

After all, a brief glance at the effects the ingestion of ~6g of BCAA had on the respective tissue levels of leucine, isoleucine and valine (figure 2, BCAA) and the other, "missing" essental amino acids (figure 2, EAA - BCAA) should suffice to understand that though BCAAs may be the necessary to trigger protein synthesis, they are yet obviously not sufficient to "build muscle" - or how else would you explain the

pronounced reduction in the concentration of the aromatic amino acids, tyrosine and phenylalanine, in both plasma and muscle as well as muscle EAA (BCAA excluded) during the recovery period

Borgenik et al. observed in their study? The scientists at least conclude that

[s]ince tyrosine and phenylalanine are neither synthesized nor degraded in skeletal muscle, reduction in the levels of these amino acid could be indicative of an improved net muscle protein balance, i.e. an enhanced rate of synthesis and/or decreased rate of breakdown [and] could  be explained by incorporation into protein.

The accrual of muscle mass (whatever that may eventually mean, cf. yesterday's installment of the Intermittent Thoughts) thusly obviously relies on the presence of all essential amino acids and not just the "branched chained holy grail" of protein synthesis, of which the current study revealed that they (BCAA ingestion) reduced the expression of MAFbx, which regulates the protein transcription factor MyoD and the eukaryotic initiation factor-3f (eIF-3f), which, in turn is of importance in the mTOR-p70S6k signaling pathway, by 30% and 50% in the resting and exercising legs, respectively.

Figure 3: Relative (compared to placebo) mTOR and p70S6K phosphorylation in response to BCAA supplementation in exercised (EX) and non-exercised (Rest) leg at different time-points before, and after single-legged leg presses (data adapted from Borgenvik. 2011)

As figure 3 finally goes to show, we see the "usual" increases in mTOR and p70S6K phosphorylation that are commonly held responsible for the downstream increases in protein synthesis, and which were obviously more pronounced in the exercised compared to the non-exercised leg. The latter may be ascribed to what the scientists cautiously label a ...

[...] tendency for BCAA supplementation to attenuate the elevation in the level of Rheb mRNA in both resting (1.7-fold under the placebo versus 1.2-fold in the BCAA condition) and exercising muscle (2.4-fold versus 1.5-fold).

This ameliorative effect on Rheb, the low-molecular weight GTPase located immediately up-stream of mTOR, in combination with the exercise induced reductions in REDD2 expression (another negative regulator of mTOR) the scientists observed in the exercised leg are actually where we are currently at, as far as our understanding of the complex protein synthetic machinery goes. It is here at the gene-level where amino acid supplementation and its effect on Rheb and exercise and its effect on REDD synergize and facilitate those muscle gains trainees have been making for years often without any understanding of the biological underpinnings.

And though we may eventually be able to squeeze out another 5-10% more muscle mass, when we eventually get the "whole picture", I seriously doubt that even the most thorough understanding of the underlying biomolecular processes will change such basic recommendations as "take your 25g of fast digesting whey as a bolus immediately post workout" (cf. "Never Sip Your Whey!") - or what would you say?

Building a Bigger Engine: Resistance After Endurance Training Increases Mitochondrial Biogenesis & Protein Synthesis and Ramps Up Fat Metabolism

Image 1: There is nothing wrong with some "classic cardio" training, especially if you spike it up to build your mitochondrial engine

In a recent review of the literature, J.M. Wilson from the University of Tampa analyzed the results of 27 studies to determine whether and to which extend concomitant endurance training does / could have detrimental effects on the outcomes of resistance training (Wilson. 2011). And I suspect that it will not surprise you that Wilson found negative correlations "between frequency (-.26 to -.35) and duration (-.29 to -.75) of endurance training [and] hypertrophy, strength, and power." What is yet also noteworthy is a similarly significant (p<0.05) correlation with lower body fat levels and maximal heart rates on part on those strength athletes who did some sort of endurance exercises. Now, a more recent study which is soon going to be published in Journal of Applied Physiology sheds some more light on the complex interplay of endurance and resistance training and the potential benefits of combining both to build a "bigger mitochondrial engine" (Sahlin. 2011).

Interestingly, the Swedish scientists started out with a diametrically opposed hypothesis. Sahlin et al. expected that the signaling of mitochondrial biogenesis, of which it is common knowledge that it is promoted by "classic" low(er) intensity endurance exercise, would be impaired by resistance exercise. To validate their hypothesis, the scientists had a group of ten healthy subjects (7 males and 3 females; age, 26 ± 1.2 (mean ± SE) yr; height, 177 ± 2.9 cm; weight, 72 ± 3.5 kg) perform either 60min of endurance exercise (65% of VO2Max on a cycle ergometer) alone (E), or in combination (R+E) with a subsequent bout of 6 sets of leg presses at workloads corresponding to 70, 75, 80, 80, 75 and 70 % of the individual 1RM with 3 min rest between each set (cf. figure 1)

Figure 1: Graphical overview of the study outline (based on Sahlin. 2011).

Muscle biopsies were taken before and after the exercise protocol, to which the subjects had been randomly assigned and which was repeated 2 weeks (4 weeks in the female participants to avoid any influence of the menstrual cycle) later with subjects from the E group performing E + R and vice versa. The results, I'll say so much, were by no means what the researchers had expected.

Figure 2: Changes in lactate and muscle glycogen content in response to endurance (E) and combined endurance and resistance (E+R) training (calculated based on Sahlin. 2011).

While there were the expected differences in lactate levels, and glycogen content of the biopsied legs (cf. figure 2), the increase in the phosphorylation of mTOR and its upstream regulator Akt (you should know these promoters of protein synthesis from the posts in the Intermittent Thoughts series and my dissertations on other studies, by now ;-) was not only exclusive to the endurance + resistance training group (E+R), it was probably also much more pronounced than one might expect with 6 sets of leg presses and lead to an almost dramatic increase in p56Sk1 phosphorylation (do I have to mention that this happened "although" the subjects trained >12h fasted and remained fasted for the whole study period?) - a relatively reliable marker for protein synthesis (cf. figure 3).

Figure 3: Changes of key enzymes envolved in the phosphorylation of key enzymes in the protein synthetic cascade in response to endurance (E) and combined endurance and resistance (E+R) training (calculated based on Sahlin. 2011).

Morover, and totally contrary to what the scientists had expected, the expression of the key enzyme for mitochondrial biogenesis and increased fatty acid oxidation, PDK4 was significantly elevated, not suppressed, in response to the additional leg training (cf.  figure 4).

Figure 4: PDK4 phosphorylation (arbitrary units) in response to endurance (E) and combined endurance and resistance (E+R) training (calculated based on Sahlin. 2011).

The research hypothesis that a (relatively short, but intense) bout of resistance training subsequent to a mitogenic "classic" cardio regimen would blunt the beneficial effects of the latter on mitochondrial biogenesis is thusly more than falsified. As it turns out, the 6% increase in total work-load due to the addition of the 6 sets of leg presses makes a huge and desirable (!) difference (way beyond what an over-simplified workload = output equation would explain) in terms of "building a bigger engine" - an engine that will keep you lean on a bulk and help you lean out while your dieting.

If you are no powerlifter, it is thus probably no mistake to keep some "classic cardiovascular" exercise in your regimen, especially if you spice it up with a subsequent short bout resistance exercise - another option, and I am repeating myself here, would obviously be a high intensity cardio session (cf. HIIT). That being said, change has time and again proven to be the key to continuous improvements in exercise performance, muscular growth and strength, to incorporate both spiced up "classic cardio" and HIIT in your routine could not only improve your results (in view of the protein synthetic response, you could even "grow" on such an E+R day), it will also prevent you from getting bored with performing the same routine day in and day out and if you asked me, that is an even more fundamental key to success than a X% increase in the phosphorylation of whatever key enzyme ;-)

Glycogen-Free Muscle Growth - Erratum: Differences in P70K-Phosphorylation Between Glycogen De- and Repleted Leg Even Less Significant Than Previously Reported.

Image 1: For the SuppVersity Super-Student Duong Nguyen, training in the semi-fasted state worked wonders. Want to know more? Read his guest-post and visit his blog.

This is post #713 and another premier. It's the first time that I have to go back (at least partly) on something I posted three weeks ago in a post about the myth that well-stocked muscle glycogen stores would be necessary to induce an anabolic growth response in skeletal muscle. Those of you who read the respective blogpost probably remember that I had to rely on the little information there was in a short abstract that had been published in the program of the 2011 ISSN conference, because a full paper with all the information on the study had (and still has) not been published.

Now, three weeks and a long and interesting email-correspondence with the author, Donny Camera from the RMIT University in Melbourne, Australia, later, I have to admit that (my interpretation of) the abstract was not completely correct. In the abstract it says (Camera. 2011):

p70S6K^Thr389 phosphorylation in LOW [glycogen depleted leg] increased in both nutrient (15-49 fold) and placebo (∼8 fold) groups 1 h and 4 h post-exercise compared to rest (P <.05) but was only different from rest 1 h post-exercise in NORM in the nutrient group (∼36 fold, P <.05).

Back then (and to be honest, even now that I know what Donny actually meant), my understanding of "but was only different from rest 1h post-exercise in NORM in the nutrient group" was that there was no additional benefit from exercise in the normal leg, unless a post-workout drink consisting of 20g Whey + 20g maltodextrin was consumed. While I found that initially surprising I assumed that the absolute p70S6K response (remember the abstract provides information about the relative changes, only) in the non-depleted leg [NORM] would have been much higher than in the glycogen depleted leg, so that the addition of a protein + carbohydrate post-workout shake would not really make a difference.

Figure 1: This graph depicting the p70k response in the glycogen depleted and the normal leg is of merely illustrative nature the data is not identical with the original material from the study (Camera. 2011), but was made up to adequately represent the most important findings.

Now, that I have the absolute data available, I see that this is not the case (cf. schematic illustration in fig. 1). The absolute p70S6K response to exercise was within the statistical margin of error identical in both groups regardless of whether post-workout nutrients were or were not supplied. Still, the addition of the whey + maltodextrine combination increased the p70S6K response in the "anabolic" 1h window roughly 4-fold above the increase that was seen with exercise alone. In that, the increase in the NORMAL leg may have been greater; more importantly, however, the maximal degree of phosphorylation at t=1h post exercise was identical in both legs. Furthermore, the absolute data underlines the aforementioned importance of post-workout nutrient supply (in this case in the form of fast digesting protein + carbohydrate sources), as the provision of the whey + maltodextrine formula quadrupled the already pronounced increase in p70S6K phosphorylation irrespective of the glycogen status of the trained leg.

Thus, it turns out the relative values from the abstract, on which I based my previous blogpost, did provide an initial impression, but not the whole picture oft what happens on the cellular level when you train a "fasted muscle" (which is why I usually do not even bother with abstracts, if I do not have access to the full text, but in this case, there simply was no fulltext, and the results were too interesting to keep them back). Contrary to the researchers initial hypothesis was there not only more than enough "gas in the tank" of the LOW leg even after selective glycogen depletion to for the protein synthetic cascade to be put into motion, the absolute degree of p70S6K phosphorylation and (this is only a reasonable assumption) exercise and nutrient induced protein (re-)synthesis were also identical.

Image 2: Glycogen stores (magenta staining) in liver cells (A. Gunin)

Did you know that the rate of glycogen depletion in the human liver upon fasting (no exercise) is about 0.3mmol/kg liver tissue per minute? If we assume that, in a fed state, the average human being stores roughly 300mmol/kg glycogen in his/her liver, a 16-hour fast, as Martin Berkhan from leangains.com suggests them, would use roughly 96% of your liver glycogen stores (calculations based on Nilsson. 1973) - probably no coincidence that we are seeing the first detrimental effects on resting energy expenditure after this time-span, what do you think?

Personally, I was not surprised by this result, partly because I still think that this way of selective glycogen depletion is not representative of whole body starvation, the one and only state of which I would assume that you would see profound decreases in the anabolic response to exercise. And since I know you would be asking: I assume you will see identical results if you train (intermittently) fasted (and Duong is the living testimony to this hypothesis ;-), although your whole body (and especially liver) glycogen levels will probably be lower than the ones of the study participants.

Maximal Protein Synthesis in the Elderly: How Much Protein Does it Take? Another Study to Suggest More is Better!

Maximal protein synthesis requires protein, but how much exactly you need will depend on your age - the older you are the more PWO protein you'll need.

Scientists from the University of Auckland were fed up with the lack of information about the differential response in protein synthesis in response to the ingestion of various amounts of protein. Accordingly, Randall F. D’Souza et al. conducted a study to characterize the changes in intramuscular levels of EAAs and BCAAs and the expression of the "protein pump" p70S6K at Thr389, a marker of protein synthesis, in response to resistance exercise and graded ingestion of whey protein in older men.

As a regular SuppVersity reader you will probably already think: "Where is the actual measurement of the fractional protein synthesis?" The unfortunate answer: It's not there.

You can learn more about protein intake at the SuppVersity

Are You Protein Wheysting?

5x More Than the FDA Allows!

Protein requ. of athletes

High EAA protein for fat loss

Fast vs. slow protein

Less Fat, More Muscle!

Previous research had show that the ingestion of graded amounts of high-quality protein such as whey after resistance will maximize with "only" 20g of egg protein (Moore. 2009) or whey (Witard. 2014) in young men. Multiple studies in older adults (>60 years), on the other hand, suggest that they exhibit a lower anabolic signaling and MPS response to protein feeding, resistance exercise, and the combination of feeding and exercise when compared to young men (Cuthbertson. 2005; Fry. 2011; Burd. 2013). Scientists call this phenomenon age-related "anabolic resistance" (Yang. 2012b).

Figure 1: In contrast to the fractional protein synthesis in the elderly, which increases with increasing amounts of protein, the FSR of young men shows a ceiling effect at 20g+ whey protein (Yang. 2012a; Moore. 2009)

As you can see in Figure 1 from a 2012 study by Yang, the same 20g of extra-whey (total dose 40g) that was useless in young men, lead to a significant increase in protein anabolism in elderly men. Compared to young men, the MPS response to feeding 40 g of protein was yet still slightly lower in older vs. count men (Yang. 2012a; Churchward Venne. 2013b).

What is particularly relevant for the study at hand, and the previously criticized absence of actual MPS measurements is the fact that deficits in feeding induced p70S6K phosphorylation may at least partially underpin anabolic resistance in aged skeletal muscle (Cuthbertson. 2005), which is why measuring the p70S6K phosphorylation in older human subjects (mean age 71 years) in response to the graded ingestion of whey protein after a leg workout consisting of three sets of 8–10 repetitions of bilateral barbell smith rack squat, 45°leg press, and seated knee extensions at 80% of the subjects' predetermined 1R is not as irrelevant at it may initially have seemed.

Workout + supplements, that's the "whey to go" ;-)

The exercises were performed in a circuit manner with 1 min rest between each exercise and 3 min rest between subsequent sets, the exercise protocol took approximately 20 min to complete. Following completion of the exercise protocol, subjects were immediately provided with a fixed-volume (350 mL) beverage, containing a flavored noncaloric placebo, or oneof the four doses of whey protein concentrate (10 g, 20 g, 30 g, or 40 g).

Figure 2: Intramuscular amino acids. This figure is a heat map which shows groups means fold changes from the resting fasted condition. Green represents a decrease in amino acid content, white represents no change, and red represents an increase in amino acid content (D’Souza. 2014)

Subjects were instructed to ingest the beverage within 2 min and were required to ingest the total volume provided. Following consumption of the supplements, subjects rested in a supine position throughout the 4 h of post-exercise recovery with additional muscle biopsy samples collected at 2 and 4 h post exercise.

Figure 3: Higher protein intake = higher increase in p70S6K phosphorylation (left graph). This increase is linearly associated with intramuscular leucine levels (right graph | both from D’Souza. 2014)

As you can see in Figure 3, there was a similar dose-dependent increase in p70S6K as it was observed previously for MPS in skeletal muscle of elderly subjects by Yang et al. (2012b). In fact, the fold change in the phosphorylation of p70S6K (Thr389) at 2 h post exercise was correlated with the dose of whey protein consumed (r =0.51,P<001) and was found to be significantly correlated with intramuscular leucine content (r =0.32,P=0.026).

Moreover, the intramuscular BCAAs, and leucine in particular, appear to be important regulators of anabolic signaling in aged human muscle during post-exercise recovery via reversal of exercise-induced declines in intramuscular BCAAs.

Suggested Read: "Protein Timing Does Matter! Yet Only in Trained Men. More Than 2x Higher Relative Protein Retention W/ Immediate vs. 6h Post Whey Consumption in Bodybuilders vs. Rookies" | read more.

Bottom line: In the absence of a young control group and actual muscle protein synthesis (MPS) measurement, the study at hand cannot finally answer the question, whether older men require higher amounts of protein than young ones to achieve maximal increases in post-workout protein synthesis, but it is at least another piece of evidence that "more helps more" - at least in the elderly.

As mentioned in other recent posts, there are yet still many confounding variables that would have to be controlled and modified as well to answer the important (?) question: "How much protein does it take to achieve maximal post-workout protein synthesis?" Which confounding factors that would be? Well, what about the training experience? The baseline muscle mass? The protein content of the diet? And so on and so forth || Comment on Facebook!

References:

• Burd, N. A., S. H. Gorissen, and L. J. van Loon. 2013.  Anabolic resistance of muscle protein synthesis with aging. Exerc. Sport Sci. Rev. 41:169–173.
• Churchward-Venne, T. A., N. A. Burd, C. J. Mitchell, D. W. West, A. Philp, G. R. Marcotte, et al. 2012. 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. 590:2751–2765.
• D'Souza, Randall F., et al. 2014. Dose‐dependent increases in p70S6K phosphorylation and intramuscular branched‐chain amino acids in older men following resistance exercise and protein intake. Physiological Reports 2.8: e12112.
• Churchward-Venne, T. A., L. Breen, and S. M. Phillips. 2013a. Alterations in human muscle protein metabolism with aging: protein and exercise as countermeasures to offset sarcopenia. BioFactors 40:199–205.
• Churchward-Venne, T. A., C. H. Murphy, T. M. Longland, and S. M. Phillips. 2013b. Role of protein and amino acids in promoting lean mass accretion with resistance exercise
  and attenuating lean mass loss during energy deficit in humans. Amino Acids 45:231–240.
• Churchward-Venne, T. A., L. Breen, D. M. Di Donato, A. J. Hector, C. J. Mitchell, D. R. Moore, et al. 2014. Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial.
  Am. J. Clin. Nutr. 99:276–286.
• Cuthbertson, D., K. Smith, J. Babraj, G. Leese, T. Waddell, P. Atherton, et al. 2005. Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle. FASEB J. 19:422–424.
• Moore, D. R., M. J. Robinson, J. L. Fry, J. E. Tang, E. I. Glover, S. B. Wilkinson, et al. 2009. Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. Am. J. Clin. Nutr. 89:161–168.
• West, D. W., and K. Baar. 2013. May the Force move you: TSC-ing the mechanical activation of mTOR. J. Physiol. 591:4369–4370.
• West, D. W., N. A. Burd, J. E. Tang, D. R. Moore, A. W. Staples, A. M. Holwerda, et al. 2009a. Elevations in ostensibly anabolic hormones with resistance exercise enhance neither training-induced muscle hypertrophy nor strength of the elbow flexors. J. Appl. Physiol. 108:60–67 .
• West, D. W., G. W. Kujbida, D. R. Moore, P. Atherton, N. A. Burd, J. P. Padzik, et al. 2009b. Resistance exercise-induced increases in putative anabolic hormones do not enhance muscle protein synthesis or intracellular signalling in young men. J. Physiol. 587:5239–5247.
• Witard, O. C., S. R. Jackman, L. Breen, K. Smith, A. Selby, and K. D. Tipton. 2014. Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise. Am. J. Clin. Nutr. 99:86–95
• Yang, Y., L. Breen, N. A. Burd, A. J. Hector, T. A. Churchward-Venne, A. R. Josse, et al. 2012a. Resistance exercise enhances myofibrillar protein synthesis with graded intakes of whey protein in older men. Br. J. Nutr. 108:1780–1788.
• Yang, Y., T. A. Churchward-Venne, N. A. Burd, L. Breen, M. A. Tarnopolsky, and S. M. Phillips. 2012b. Myofibrillar protein synthesis following ingestion of soy protein isolate at rest and after resistance exercise in elderly men. Nutr. Metab. 9:57.

Well-Stocked Muscle Glycogen Stores Not Necessary For Exercise Induced Muscle Anabolic Response. Additional 5x Increment by Post(!)-Workout Whey + Cho Supplement.

Image 1: Glycogen depleted or not,
post-workout protein, preferably from a 
leucine-rich, fast digesting and nutritionally
complete source such as whey, is a must.

It is one thing that many trainees feel they perform better, train harder or have better endurance, when they (over-)"load" their muscle glycogen stores pre-workout. And as long as their need for carbohydrates is not merely imaginary, i.e. they feel sluggish and their gym performance sucks, whenever they are training on empty glycogen stores, I am quite sure that they will also make better gains. This mechanism would yet be completely different from any immediate, yet hitherto scientifically not validated, facilitative biomolecular effect of well-stocked glycogen stores on muscular hypertrophy, as it is proposed by many advocates of preworkout or even 24/7 carbohydrate (re-)feeding.

Dr. Connelly, who talked about this issue at length in the past installments of the BodyRX Show, was kind enough to remind me that back in 2007 Coffey et al. from Stuart Phillips' group at McMaster University, in Hamilton, Ontario (Canada), conducted a study that was based on an antithetical hypothesis, i.e. whether or not commencing resistance exercise with low muscle glycogen would enhance the encoding of genes implicated in muscular hypertrophy (Coffey. 2007). Yet, while there were significant differences at rest for the glycogen depleted vs. the normal leg of the subjects, both the increased GLUT4-MRNA expression, which is a sign of an increased capacity for glucose uptake, as well as the reduced expression of atrophic atrogenes (responsible for proteolysis, i.e. protein degradation) were overridden by exercise. Now, four years later Donny Camera from the University of Melbourne presented the results of a recent colloberation with the scientists from McMaster at the American College of Sports Medicine Conference in Denver, this year (Camera. 2011). The intention of this 2nd study was to elucidate the "effect of divergent glycogen content and subsequent post-exercise nutrition on anabolic signaling target p70S6 kinase during the early recovery period" after the completion of a standardized resistance training protocol.

Illustration 1: Very simplified illustration
of the role of mTOR and p90S6K
in protein synthesis.

Did you know that p70S6 kinase is a key component of the mTOR (the mammalian target of rapamycin) signaling cascade? The activation of mTOR via branched chain amino acids (leucine in particular) has been shown to increase p70S6K phosphorylation (the phosphorylation is equivalent to 'switching' it on). In a similar vein, physical exercise can activate protein synthesis via phosphorylation (activation) of p70S6K. The degree / increase / decrease of p70S6K kinase phosphorylation is thus considered a reliable indicator of the protein anabolic response to supplement and exercise protocols.

The evening before the actual experiment was conducted, the 16 resistance-trained male subjects (~23y) who participated in the study, reported to the laboratory in order to perform a single-legged cycling exercise to fatigue. In order not to upset the thusly established difference in glyocogen content between the trained (LOW) and the untrained leg (NORMAL), the subjects consumed an identical low carbohydrate meal after the workout and had to abstain from foods until the subsequent day, when they performed 5 unilateral leg press repetitions at 80% of their personal 1RM (one-repetition-max) with both their normal, as well as the glycogen depleted (LOW) leg. Muscle biopsies were taken 1h post exercise, and subjects consumed either a 0.5l post-workout shake that consisted of 20g whey + 40g maltodextrin or placebo immediately post and 2h after the exercise regimen.

Figure 1: Increase in  p70S6K phosphorylation in 16 resistance trained males after unilateral leg press exercise in normal and glycogen depleted leg relative to baseline (data adapted from Camera. 2011)

Although the muscle glycogen content increased exclusively in the nutrient (20g whey + 40g maltodextrin) group, significant increases of phosphorylation of p70S6K one of the key regulators of protein synthesis were seen in both legs of the subjects. As my plot of the restricted data I could extract from the abstract in the conference protocol (a paper obviously has not been published, yet) indicates, this increase was augmented up to 5x in the 1-4h hour post workout window in the glycogen depleted leg. While there was still a 8x increase in p70S6K phosphorylation in the glycogen-depleted leg even in the absence of post-workout nutrient repletion, post-workout nutrient (re-)feeding turned out to be necessary to illicit any increase in p70S6K phosphorylation over baseline in the normal leg.

Note that the baseline levels of the LOW and the NORMAL leg were probably different and the 8x increase could thus have lead to an absolute level of p70S6K phosphorylation that was still lower than in the NORMAL leg..

These results do not only contradict the initially raised hypothesis that well-stocked glycogen stores would be a necessary or at least facilitative prerequisite for the muscle anabolic response to exercise to take place, they also (re-)raise the question whether "training on empty" may not after all be advantageous if ...

1. the training performance is not effected by the lack of muscle glycogen and
2. the muscle anabolic response is augmented via appropriate post-workout nutrient-replenishment

Since this conjecture is yet solely based on the relative increases in phosphorylation, the scientists cite in their abstract, it is far from being a valid scientific hypothesis. We will probably have to wait for the publication of a respective paper (or ask someone who was lucky enough to attend the presentation for the absolute values; cf. "Note...", above), to get a preliminary answer on any beneficial effect exercising in a glycogen depleted state could have. In that, I would like to add that its artificial incarnation, i.e. the induction of local glycogen depletion, as it was practiced in the study at hand, has no significance with regard to the whole body (including liver) glycogen depletion some trainees experience as a result of (over-)training and no-carb (over-)dieting. In case of the latter, it does not take a rocket scientist to be able to tell that this won't have any beneficial effect on the gains people are making in the gym.

Can You Build Muscle By Depriving it of Glucose? Muscle Cells Increase Protein Synthesis After Only 3 Minutes of Running on Empty, But Beware of Jumping to Conclusions

Unless you're already infected with carbophobia and the "oh god, I got to not eat after a workout to maximize growth hormone"-bullshit-viruses, it sounds awkward that glucose deprivation should increase protein synthesis, but in the end, it all depends on the right circumstances

It may sound stupid and certainly not like something you would read on the SuppVersity, one of the few places on the Internet that has not been infected with "carbophobia", yet, but it's actually exactly what a recent study from the , University of Napoli "Federico II" suggests: Muscular glucose deprivation promotes the activation of mTOR signaling pathway and will thus increase protein synthesis!

And no, we are not talking about even more cognitive masturbation and theoretical considerations, here. The conclusions Maria Concetta Miniaci and her colleagues present in the latest issue of Pflugers Arch - Eur J Physiol are in fact based on experimental evidence - experimental evidence that is, if you think it through - not contradicting its practical counterpart!

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Evidence from a rodent in vitro study, but evidence of which not just Miniaci et al. (2014) but also I am confirmed that it is relevant for humans, as well.

In a way you may say that Miniaci et al. were inspired by the increased interest in the pro-anabolic effects of blood flow restriction. Corresponding studies using aterial and venous compression, induced by either muscle contraction at high intensity or by pressure cuff will, as the scientists point out, induced "short reduction of blood flow and therefore a reduced delivery of nutrients to the muscle" (Barcroft. 1939; Schwartz. 2000).

"Since glucose is an important energy substrate for skeletal muscle, we presumed that a brief period of glucose deprivation may trigger, as metabolic stress, the anabolic signaling cascade leading to an increased rate of protein synthesis when blood flow recovers and all nutrients are again available (Holloszy. 1996)." (Miniaci. 2014)

To test this hypothesis, the scientists investigated the effect of glucose deprivation on mTOR signaling and protein translation in L6 cell line, "a well established in vitro model system for studying skeletal muscle physiology" (Miniaci. 2014); and their results demonstrate that glucose deprivation upregulates mTOR and its downstream target the ribosomal S6 kinase (p70S6K), through the activation of NO/PI3K/Akt/mTOR/p70S6K signaling pathway.

Figure 1: Glucose deprived and thus stressed muscle cells increase mTOR and p70S6K and show an increase in protein synthesis after only a short time of "running on empty" (Miniaci. 2014)

Now, the interesting thing is that the observed results are pretty similar to what we see in response to regular resistance training, as well. The "pump induced" occlusion of the regular nutrient flow, as well as the acute (!) glucose deprivation in response to exercise could thus be more than casually related to the post-workout increase in protein synthesis. They could, and this is also what the scientists believe, actually be mechanistically involved in the post-workout increase in protein transport into the skeletal muscle.

mTOR, AMPK, NOS and good (=EU-)stress

In that, auxiliary data from the study at hand showed that the post-workout increase in protein synthesis is directly related to an increase in (surpris!) nitric oxide synthesase activtiy (this is the stuff that is not increased by the mere provision of the NOS substrate arginine, so that corresponding pump-supplements pretty useless). As the Italian researchers point out, NOS is activated in L6 cells exposed to glucose deprivation. Why this is the case, however is still unclear.

"[It] is likely that an increase in AMPK activity, in response to energy depletion, can account for activation of NOS. Indeed, NOS and AMPK can interact through a positive feedback loop. In particular, AMPK has been found to phosphorylate and activate skeletal muscle NOS inducing an increase in glucose uptake." (Miniaci. 2014)

If AMPK is in fact the main link, here, the findings of the study at hand, would - just like previous studies on exercise induced increases in glucose uptake lead us back to one surprisingly positive mechanism, which triggers both, glucose uptake and protein synthesis: Metabolic stress! In this case, glucose starvation and susequent acute increases in AMPK expression during exercise.

Figure 2: Urine urea levels after working out in a glycogen replete (CHOH) vs. depleted (CHOD) state (Mullin. 1980)

What is important, though, is that you understand that this is an acute phenomenon - a beneficial effect of short-term glucose deprivation and not a result of running around glycogen depleted 24/7. The latter would not just decrease your training performance significantly. Studies by Lemon & Mullin (1980), for example, show that working out in a glycogen depleted state is associated with an almost 3x increase in markers of protein loss (urea; see Figure 2) compared to the same workout in a glycogen replete state.

More recent studies by Howarth et al. which measured the rate of leucine oxidation and other markers of protein catabolism confirm these results (Howarth. 2009), where the net amino acid loss doubled and underline that the last thing you want is to do if you're training for size gains and/or lean mass retention is to train in a glycogen depleted state.

Rather than that, I suggest you take another look at my glyocogen repletion recommendation from a previous article, hit the gym with full glycogen stores and see as the AMPK and NOS expression increases to trigger an increase in glucose uptake and protein synthesis while the glycogen stores are fading during your workout. Remember: It's about cycling high and low levels of energy availability and you certainly don't want to stay on the low end side for more than the few minutes it may take to trigger increases in protein synthesis.

Reference:

• Barcroft H, Millen JL (1939) The blood flow through muscle during sustained contraction. J Physiol 97:17–3.
• Holloszy JO, Kohrt WM (1996) Regulation of carbohydrate and fat metabolism during and after exercise. Annu Rev Nutr 16:121–138 
• Howarth K, et al (2009). Effect of glycogen availability on human skeletal muscle protein turnover during exercise and recovery. Journal of Applied Physiology 17:19.
• Lemon, PW, Mullin JP (1980) Effect of initial muscle glycogen levels on protein catabolism during exercise. Journal of Applied Physiology 48:624-629.
• Miniaci, M. C., Dattolo, M. G., Irace, C., Capuozzo, A., Santamaria, R., & Scotto, P. (2014). Glucose deprivation promotes activation of mTOR signaling pathway and protein synthesis in rat skeletal muscle cells. Pflügers Archiv-European Journal of Physiology, 1-10.
• Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG (2000) Central nervous system control of food intake. Nature 404:661–671.

Exercise Velocity Does not Determine Hypertrophy Signaling in Eccentric Exercises: Akt, mTOR, P70s6k Protein Phosphorylation Identical for Fast and Slow Movements

Finally, a study (Roschel. 2011) related not only to nutrition and supplementation for exercise, but to exercise itself! The respective paper was published in the Applied Physiology, Nutrition, and Metabolism on April, 13th, and reports the results of a study on the effects of exercise velocity on markers of muscle hypertrophy, specifically, Akt/mTOR/p70^s6k.

Figure 1: Schematic illustration of the mTOR signaling cascade (Betz, Charles. Wikipedia)

Roschel, et al. had 20 subjects, "not enrolled in any form of strength training for at least 6 months prior to the
study and without any history of musculoskeletal disorders" perform 5 sets of 8 repetitions of an eccentric knee extension exercise at either a slow (20°·s^–1; ES) or fast execution speed (210°·s^–1; EF). After the workout, biopsies were taken from vastus lateralis at three timepoints: baseline (B), immediately after (T1), and 2 h after (T2). The results did not confirm the scientists' working hypothesis that execution velocity (and thus muscle tension) would have a direct influence on Akt, mTOR, and p70^S6K expression in the trained muscles:

Akt, mTOR, and p70^S6K total protein were similar between groups, and did not change postintervention. Further, Akt and p70^S6K protein phosphorylation were higher at T2 than at B for ES and EF. MGF messenger RNA was similar between groups, and only significantly higher at T2 than at B in ES.

So, with respect to the measured variables and in the confounding case of eccentric exercises, it does not matter whether you whack your reps out at maximum speed (assuming you still maintain adequate form) or try to slow the movement down deliberately. I guess this, aside from their (ab-)use certain "supplements", is why Coleman and Co. grew monstrous muscles despite training with the worst form you could probably think of.

Remember though, the results could be completely different for concentric exercises, like pressing movements or for equal times under tension, meaning 10x more repetitions in the EF group to make up for the total time difference. Results from previous studies, such as Farthing and Chilibeck (2003) and Shepstone et al., for example suggest that compared to slow training a faster (yet still controlled) exercise execution is linked to greater hypertrophy of the elbow flexor, in general, and the biceps' type IIa (+16%) and type IIx (+18%), in particular. I guess future studies will further elucidate the exact mechanisms and guess what: the SuppVersity is where you will read about them, first!

The Anabolic Effects of HIIT: 3x30s High Intensity Intervals Increase mTOR & Ramp Up Marker of Protein Synthesis by +43% in Men and +222% in Women - Even in a Fasted State!

Image 1: While the study at hand clearly shows that HIIT, even done on an empty stomach, is anabolic, not catabolic, it appears as if women respond better to sprint exercises than men. And this assumption is not based on gene-assays but dates back to the results of a 1999 study by Esbjörnsson which showed a more pronounced CSA increase in the leg muscles of female subjects.

Not all too long ago, the general accepted consensus was that anyone whose main interest is in building muscle must abstain from any strenuous cardiovascular exercise... running on a treadmill? God-forbid! You could lose muscle. Over the last two years or so, this paradigm has began to totter, though. And now, at the beginning of 2012 I would estimate that the number of (recognized) trainers and trainees who recommend doing high intensity interval training (HIIT), if not for general conditioning, then at least as a means to shed fat, initially surpasses the number of the conventionalists who maintain that "classic cardio" training in the "fat-burning zone" was the way to go. Now, if this is not your first visit here at the SuppVersity, you should be aware that the latest scientific research supports the arguments of the advocates of HIIT. And not so much to my, as to the surprise of some researchers, this holds true not only for already well-conditioned gymrats and athletes, who want to finally pass beyond the 10% body-fat barrier, but also for the obese and metabolically deranged diabetic, who is trying to get his blood sugar under control (cf. "Hitting Diabetes With A Hammer").

The advantages of HIIT reach well beyond fat loss, but...


Moreover, a 2011 study by Naito et al., the results of which I have discussed in November 2011, shortly after it was published in Acta Physiologica (cf. "HIT Your Satellite Cells to Increase Your Gains"), already hinted at the fact that the advantages of HIIT reach well beyond its fat-burning effects. Yet although the increase in both satellite cell count and incorporation into the muscle Naito et al. observed speak for themselves, there's still rumors going round that this training style could be catabolic. In that the argument usually revolves around the notion that muscle damage is a major driving force of satellite cell recruitement and that if the latter is a necessary consequence of HIIT it would counter your efforts to build muscle. Now, aside from the fact that this argument is intrinsically flawed (I mean, what to you do in the gym, when you weight train? You break down muscle tissue!), a recently published study from the famous Karolinska Institute in Stockholm, Sweden, attests to the fact that the exact opposite is the case.

... it appears as if women could derive even greater benefit from all-out sprinting than men

Image 2: The exercise stimulus in the study was a Wingate test, one of standard procedures in exercise science.

In an earlier study from 1999 Esbjörnsson and his / her colleagues had observed that the cross-sectional area of the leg muscles of women exhibited a more pronounced hypertrophy response to sprint training than those of their male peers (Esbjörnsson. 1999). With the advent of our advanced understanding of the underlying principles of skeletal muscle hypertrophy and the central, but as those of you who read the Hypertrophy 101 know, in the current discussion possibly overemphasized position of the mammalian target of rapamycin (mTOR), Esbjörnsson et al. did now set out to examine, whether a sex-specific response of mTOR and its downstream targets could explain their previous results (Esbjörnsson. 2012).

To this ends, the scientists recruited nine men and eight women who despite participating in leasure time sports were only "in good shape" and not considered to be athletes. For the experiment the subjects reported to the lab fasted and, after a brief 1min warm-up, performed the well-known Wingate-test, which consists of three consecutive 30s all-out sprints with 20min breaks between the intervals on a braked cycle ergometer (average peak power was ~645W and ~935W for women and men, respectively, on a per-lean body-mass base, the peak and mean power was yet identical)

Figure 1: Illustration of the experimental protocol used in the study.

Before the warm-up and 140min after the 3rd sprint, Esbjörnsson et al. took muscle biopsies from the quadriceps muscles of the subjects to assess the local expression of mTOR and its downstream targets.

Figure 2: Phosphorylated AKT, mTOR, p70S6K and rpS6 (a.u.) in male and female study participants before the first and 140min after the third sprint of the Wingate test (data adapted from Esbjörnsson. 2012)

If you are not well-versed in the the intricacies of the mTOR-cascade, it appears as if the data in figure 2 would disprove the scientists' research hypothesis that "mTOR signalling is more pronounced in women than in men". After all, the increase in phosphorylated mTOR (p-mTOR) and AKT (p-AKT) in response to the three 30s seconds sprints was obviously more pronounced in the male, than in the female participants (mTOR +26% and AKT +17% greater increases; a difference which did not reach statistical significance, though).

Do women just make better use of the same stimulus?

As far as the phosphorylation of p70S6K, of which the current scientific evidence suggests that is a more appropriate measure of the "real-world" protein synthetic effect of mTOR, a completely different picture emerges. While the +43% increase in the male subjects is just about statistically significant (p = 0.04), the +222% increase in p-p70S6K in the female subjects appears to confirm what Esbjörnsson et al. already  had suspected.

Figure 3: Serum leucine and growth hormone levels at rest and after the sprints (data adapted from Esbjörnsson. 2012)

The slightly greater disappearance of leucine from the skeletal muscle of the male subjects (cf. figure 3) is yet only one of three possible explanations (and one you could counter by ingesting BCAAs, for example) Esbjörnsson et al. come up with based on the results of previous studies:

Image 3: If you look at the leg muscles
of some of the female speed skaters,
it is quite obvious that the leg muscles
of women respond pretty well to short,
intense bouts of all-out sprinting.
(the image shows Claudia Pechstein)

1. Lower accumulation of lactate and ammonia and a faster recovery of ATP levels in type II fibers of women than men
    
2. Lower levels of plasma catecholamins (=stress hormones) in response to sprint exercises in women than in men
    
3. Slower disappearance of leucine and thusly more sustained elevation of protein synthesis in women than in men

Whether it is any of these, or a combination of all three factors which is responsible for the differential response to statistically (!) not significantly different activations of mTOR and p-AKT, cannot be decided based on the available data.

An alternative explanation, which would, by the way, have real-world implications for the training practice, is (and I prefer to cite this, to avoid being accused of sexism) that...

women do not exhaust themselves as much as men during each bout of exercise and thereby elicit a smaller activation of AMPK, resulting in less inhibition of mTOR.

In view of the fact that previous studies by Esbjörnsson et al. refute this hypothesis, it appears unlike that an "over-expression" of AMPK, of which I have discussed in one of the previous installments of the Intermittent Thoughts that its locally expressed alpha-2 isoform does not inhibit the exercise induced increase in protein synthesis, anyway, could explain why similar exercise stimuli (peak and mean power per fat-free mass were virtually identical for men and women) and within the statistical margin identical mTOR responses induce a more pronounced protein synthetic response in women than in men. And whether the early(-ier) rise in serum growth hormone, which is the last possible explanation the scientists mention, has anything to do with it appears questionably, as well. After all, the data in figure 3 shows quite clearly that the overall GH response was much more pronounced in the male than the female participants.

We don't know about aliens, but for earthlings HIIT is anabolic - regardless of their sex

In essence, it does not even really matter, what the underlying cause of the sex-specific response to sprint training is. As far as I am concerned, the most significant result of the study is not the gender-difference, but the simple, yet as the scientists point out "novel" finding that "repeated 30-s all-out bouts of sprint exercise, separated by 20 min of rest, increased Akt- mTOR signalling in skeletal muscle." And this effect was observed in both men and women. Now, this is allegedly not exactly your "usual" HIIT protocol, if you do yet take into consideration that it was performed after an overnight fast and went without BCAAs, protein shakes all the other "obligatory" anti-catabolics, the average gymrat uses to avoid the purported catabolic effects of high intensity conditioning work, I would dare to say that it HIITs another (if not a final) nail into the lid of the casket of the "HIIT = catabolic" myth.