Protein Wheysting?! No Significant Increase in PWO Protein Synthesis W/ 40g vs. 20g Whey, But 100% Higher Insulin, 340% More Urea & 52x Higher Oxidative Amino Acid "Loss"

No, I don't think the results would have been different, if the subjects had been young women. For older guys and gals, on the other hand, I am not 100% sure.

It has been a while since we've been taking a look at one of the two or three dozen "whey increases muscle protein synthesis" studies and, officially, we would have to wait not just for Santa, but actually until January 2014 to take a glimpse at the results Oliver C Witard, Sarah R Jackman, Leigh Breen, Kenneth Smith, Anna Selby, and Kevin D Tipton present in their soon-to-be-published paper in the journal of the American Society for Nutrition (Witard. 2014).

The intention of the researchers was (yet again) to "characterize the dose-response relation of postabsorptive rates of myofibrillar MPS to increasing amounts of whey protein at rest and after exercise in resistance-trained, young men", (Witard. 2014). This is nothing new, but still right up the average SuppVersity reader's alley, I suppose.

So what about the study design

The design of the study was simple. The 48 healthy volunteers consumed a standardized, high-protein
(0.54 g/kg body mass) breakfast. Three hours later, they all performed a standardized bout of unilateral exercise, consisting of 8x10 leg presses and leg extensions at 80% of their individual, predetermined one-repetition maximum. "Immediately" (max. 10min) after they were done with the leg workout the volunteers consumed

• 0g, 10g, 20g, or 40g whey protein isolate

as a post-workout protein shake, of which I don't have to tell you that it was likewise... standardized, right! The subjects were then hooked up with the necessary instruments and tools to measure their

• postabsorbtive rates of myofibrillar protein synthesis (MPS) , 
• whole-body rates of phenylalanine oxidation and 
• urea production 

over a 4-h period (the stopwatch started ticking the very moment the subjects had ingested the protein shake) in all four arms of this parallel research design, single-blind study with 7 subjects in each of the 0, 10, 20, and 40g whey protein isolate groups.

Change (%) in myofibrillar and sarcoplasmic protein synthesis after ingestion of 25g whey at rest (FED) and resistance exercise (FED-EX) after 3h and 5h (Moore. 2009a)

Just a reminder: You do remember that there is another muscular compartment where we can measure protein synthesis? Right? The sarcoplasma, i.e. the zone around the myofibers, where the satellite cells reside. At least for the exercised leg, in the study at hand, this may not be that important, though, because "in contrast [to protein feeding at rest], resistance exercise rapidly stimulates and sustains the synthesis of only the myofibrillar protein fraction after protein ingestion" (Moore. 2009; my emphasis). The word "only" is slightly misplaced. If you look at the figure on the left, it's obvious that "mainly" or "more significantly", would probably be more accurate.

Now that you know all the important details about the study design, it's almost time to take a look at the results. Before we finally do that, let's just briefly recapitulate the results of (Cuthbertson. 2005) who observed that 10 g EAAs at rest and (Moore. 2009b) who observed that 20 g egg protein after exercise were "optimal for the maximal stimulation of MPS in young adults". This is after all, what the researchers hypothesis that "20 g of whey protein (~10 g EAAs) would be sufficient for the maximal stimulation of myofibrillar-MPS rates at rest and after resistance exercise in trained, young men" (Witard. 2014) was based on.

Figure 1: Post-exercise serum insulin (AUC, µmol/ml x 4h) and leucine peak (mmol/ml), total phenylalanine oxidation (AUC µmol/ml x 4h x 100), urea production (AUC µmol x 4h) and plasma urea (AUC mmol/l x 4h), as well as myofibrillar protein synthesis (MPS) in the 4h after the workout (Witard. 2014).

As you can see, the actual study results confirm the scientists suspicion: The 20g of whey protein did maximized the myofibrillar protein synthetic response to a hypertophy-oriented leg training workout (see bottom line for an explanation of why I chose to underline the word "leg") in rested and exercised muscle of ~80-kg resistance-trained, young men.

We are talking about statistical significance here: I know what you are going to tell me, now. And yes, you are right. The protein synthesis was in fact higher, but that's more of a matter of how sustained the increase was and not a matter of a "faster" protein synthesis. In other words, with 20g of a fast absorbing whey protein and a whole meal with slow absorbing proteins 30-40min after you will achieve the same - if not higher muscle protein synthesis rates in the long(er) run (>2h)... ah, and by the way: The response in the untrained leg confirms: There is not additional MPS stimulus from 40g vs. 20g of whey (much contrary to the insulin spike, by the way ;-).

The side-finding that this in medical terms "high" amount of whey also lead to significant increases in urea production is - at least in my humble opinion not surprising. The increased ammonia production due to higher protein oxidation rates does after all have to be cleared from the body. Against the background that this process is facilitated by the urea cycle, anything but the observed increase in urea production would have been startling.

"Confirmed: All Wheys, Not Just Hydro Whey Boost Glucose Uptake and Liver + Muscle Glycogen Supercompensation. Plus: How Can Taurine help?" | more

The fact that this increase in urea production and plasma concentrations did occur in the first place, on the other hand, is a clear cut sign for the onset of "wastefulness" with higher protein consumption - or as Selby et al. put it:

"Indeed, in the current study, urea production rates , as well as plasma urea concentrations, were markedly raised with the ingestion of 40 g protein.

Thus, instead of incorporation into muscle protein, the metabolic fate of excess exogenous amino acids contained in the 40WP was predominantly the oxidation or excretion as an indication that a state of amino acid excess was reached." (Selby. 2014)

Whether you consider this a "waste" of valuable dietary protein or not is probably a matter of your personal concept of protein nutrition.

If you are on the "protein worshipper" side of the devide, you will probably argue that you better "burn" protein for energy than carbs or fats, because otherwise you would have to eat less protein and  more carbohydrates + fat and would "become fat". It goes without saying that this is bullshit - not to mention that anyone who is interested in performance and the sanity of his doctor. The poor guy would freak out, when he'd see the elevated AST and ALT levels the combination of "protein only" diets + intense physical exercise are going to produce.

Your doctor's mental sanity or the excited calls of his receptionist are probably not really your concern, but I would still not discard the performance and, in the long run, metabolic and psychological detriments from running on protein only. From an (bio-)energetic perspective it's the least effective of the three macronutrients and thus not exactly a suitable fuel source for high performance athletes.

"So what would you put into a post-workout shake, Adel?" Personally, I have ~30g of whey protein and some fruit, like 1-2 bananas, a ton of water melon, or whatever else I have lying around. If no fresh fruit is available, I just grab some instant oats. And while I know that the carbs won't help with protein synthesis (Koopman. 2007), there is hardly any better timepoint to use the massive isulin spike and shuttle the glucose into the muscle than after the workout (van Loon. 2000). For me personally, the addition of carbs also prevents the brainfog, I get due to low blood sugar after an intense leg-workout and a protein shake without carbs. So, if you feel like you're not thinking straight or would have to go to bed after your shake, I would try to fix that by adding some carbs to the equation.

Bottom line: With the study at hand we (will) get further confirmation of the existence of a protein threshold of ~20g of whey protein, beyond which we won't see additional increases in acute myofibrillar protein synthesis after having a high protein breakfast and the completion of a standardized hypertrophy-oriented leg workout in young, healthy, male individuals.

If you wonder about the many underlined words in this conclusion, I may remind you of the fact that all these words describe boundary conditions that won't be fulfilled for everyone: There are more than enough people who don't have a high protein breakfast. There are people who train their whole body in a single session and would thus upregulate the protein synthesis in more than just the leg muscles. Not everyone is still young (and there is albeit inconclusive evidence that older individuals need more protein). For long-term muscle gains the sarcoplasmic protein synthesis may and the long-term (not acute) net protein balance definitely is more important than the acute increase... I could go on, but I guess you see, where this is heading: Theoretically, we'd have to do another 100 studies, but I am not sure whether Glaxosmith Kline who support Tiptons research would want to finance all of these ;-)

Reference:

• Cuthbertson, D., Smith, K., Babraj, J., Leese, G., Waddell, T., Atherton, P., ... & Rennie, M. J. (2005). Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle. The FASEB journal, 19(3), 422-424.
• Koopman, R., Beelen, M., Stellingwerff, T., Pennings, B., Saris, W. H., Kies, A. K., ... & Van Loon, L. J. (2007). Coingestion of carbohydrate with protein does not further augment postexercise muscle protein synthesis. American Journal of Physiology-Endocrinology And Metabolism, 293(3), E833-E842.
• Moore, D. R., Tang, J. E., Burd, N. A., Rerecich, T., Tarnopolsky, M. A., & Phillips, S. M. (2009a). Differential stimulation of myofibrillar and sarcoplasmic protein synthesis with protein ingestion at rest and after resistance exercise. The Journal of physiology, 587(4), 897-904.
• Moore, D. R., Robinson, M. J., Fry, J. L., Tang, J. E., Glover, E. I., Wilkinson, S. B., ... & Phillips, S. M. (2009b). Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. The American journal of clinical nutrition, 89(1), 161-168.
• van Loon, L. J., Saris, W. H., Kruijshoop, M., & Wagenmakers, A. J. (2000). Maximizing postexercise muscle glycogen synthesis: carbohydrate supplementation and the application of amino acid or protein hydrolysate mixtures. The American journal of clinical nutrition, 72(1), 106-111.

45x More Testosterone Yet Identical Increase in Protein Synthesis: MPS Response to Exercise + 25g Whey in Men vs. Women Challenges Common Wisdom About Androgens

Image 1: Is it not testosterone that makes the difference?

It is an open secret that women are having a much harder time building muscle than men, and it is another instance of (bro-)scientific wisdom that the obvious lack of testosterone in female strength athletes would be the underlying reason. Right from Stuart M. Phillips lab at the Department of Kinesiology of the McMaster University in Hamilton, Ontario, Canada, comes a new study (West. 2012) which puts yet another questionmark behind the anabolic prowess of testosterone (if you still believe that a transient increase in testosterone will help you build muscle, I suggest you read up on "The Big T" in the Intermittent Thoughts on Building Muscle).

Women are different, but it's not about protein synthesis

In the recently conducted trial Daniel W.D. West, who has also been the lead author of the "Never Sip Your Whey" study, I covered back in November 2011, undertook another attempt to identify the intricate endo- and paracrine mechanisms of skeletal muscle hypertrophy and its sex-specific variability. To this ends, West et al. recruited 5 male and 5 female subjects, who "who were habitually engaging in two to five sessions of physical activity per week including", yet did not train legs more than twice a week.

Image 1: In this case, testosterone took a backseat, as well. With synthol, protein synthesis is yet unnecessary anyway.

Note: The selection of advanced trainees as study participants is the first huge plus of this study. After all, we all know that the exercise induced hypertrophy response diminishes with training and those of you who read the whole Intermittent Thoughts on Building Muscle series will also be aware that the protein synthetic response is limited by the maximal domain size. Further growth thusly requires restructuring / the recruitment of satellite cells and installment of new myonuclei (cf. "Growing Beyond Temporary Physiological Limits"), a time-consuming and complex process which is probably one of the underlying reason for the "growth difference" between beginners and advanced strength athletes.

On the day of the experiment, the study participants, who had consumed a standardized diet containing 15% fat, 30% protein and 55% carbohydrates (the macronutrient ratio was adapted to their habitual diets) on the previous day, reported to the lab at 6am. After the infusion of the tracer that is necessary to evaluate the protein flux and an initial biopsy, all subjects performed a bout of  intense, high-volume lower body exercise consisting of

• 5 sets of 10 repetitions of leg press at ~90% of their individual 10RM, and
• 3 super-sets of 12 repetitions of leg extension/leg curl at ~90% of 12 RM

The rest intervals between the sets were 60s, so that the whole workout should not have lasted longer than max. 20min. Directly thereafter, the subjects consumed the "obligatory" (for Phillips lab this has in fact become obligatory ;-) 25g of whey protein from the usual New Zelandian source, Phillips et al. used in all their previous study (as ridiculous as this may sound but this is a nice means of standardization ;-) and rested in a supine position for the rest of the trial. Biopsies were taken and the subjects who were sent home with a launch packet consisting of their standardized meals had to report back to the lab on the following morning for another three biopsies 24h, 26h and 28h after the test workout (the subjects remained fasted and received another 25g of whey 26h post, i.e. before the last four blood samples were drawn and the last biopsy at 28h post was performed).

Figure 1: Serum testosterone levels (in nM) and myofibrillar fractional protein synthesis rate (in %/h) before and after the resistance workout, as well as on the morning and at noon of the 2nd day (data adapted from West. 2012)

As the data in figure 1 shows, the (expected) huge difference in both basal as well as exercise induced increases in circulating androgen levels (45-fold in men vs. women) had no (not even a statistically non-significant) beneficial impact on the exercise induced increase in protein synthesis in the 28h window of opportunity (cf. "Opening the 'Anabolic Barn Door' with the Key of Science").

Akt Ser473 phosphorylation increased at 1h ( P < 0.001, main effect for time) and to a greater extent in men (sex × time interaction, P = 0.018). Phosphorylation of mTOR Ser2448 was increased at 1, 3 and 5 h (P < 0.001; Figure 4B); there was a main effect for sex (men > women, P = 0.003). Phosphorylation of mTOR Ser2448 was elevated similarly between sexes after next-day protein feeding, approximately 26 h after the exercise bout (sex  × time interaction,  P = 0.49; main effect for time, 28 > 26 h,  P = 0.006).  Phosphorylation of p70S6K1 Thr389 increased at 1, 3 and 5 h (all  P < 0.001; sex × tim e interaction,  P = 0.13) and there was a significant interaction with next-day feeding (28 > 26 h in women only, sex × time interaction,  P = 0.016; data not shown). Androgen receptor content was greater overall in men (P = 0.049) but there was no significant interaction ( P = 0.47).  

The greater increase in mTOR and Akt (both hitherto regarded as the "gas pedals" of the skeletal muscle protein synthetic machinery) are not only less pronounced, than one would expect if there was a direct interaction with testosterone levels, they also lack real world significance. After all, the area under the myofibrillar protein synthesis curve (a measure for the total protein synthetic response to exercise) was identical in the 1-5h period right after the exercise and - although West et al. did not include the respective data in their article - I would suspect that the data from the subsequent day (cf. figure 1, right) would even suggest that it must have been slightly greater in the female participants.

Testosterone useless and mTOR and Akt unreliable indicators at best?

Now, which conclusions shall be drawn from these results? Is testosterone useless? Does it not contribute to the overall greater muscle mass in men compared to women? It stands to reason that this conclusion would be about as flawed as the notion that testosterone alone would suffice to build muscle. Rather than its "inefficiency" in building muscle, this study only shows that its importance in relation to the exercise-induced increase in protein synthesis is probably way overrated.

A similar point could be made for mTOR and Akt, as well, though. Or as West et al. put it in their discussion of the results and the respective implications for future studies:

In light of this disconnect, it is worth recognizing that the phosphorylation of signalling proteins is a temporal snapshot of the propagated signal for translation initiation. It is also unclear if there is a minimum threshold signal required to initiate and completely activate or  ‘turn on’ translation. If  there is such a threshold then it seems plausible that greater phosphorylation above such a  threshold would be unlikely to further amplify the signal/lead to increased rates of translation.

For a physicist or anybody who knows a thing about "energy levels" the existence of "threshold" levels in processes taking place at a molecular level should not come as a surprise.

I suspect, we are still missing the boat with our focus on protein synthesis alone

Another question, I have been hinting at in many of my previous blogposts on the insightful studies from Stuart Phillips lab at the McMaster University, is yet whether or not the acute increase in protein synthesis (alone) is actually an acceptable predictor of skeletal muscle hypertrophy, a process which, as I have explained in detail in the Intermittent Thoughts on Building Muscle is only partly mediated by the simple accrual of amino acid chains (=proteins) within existing myofibrillar domains.

Figure 2: Graphical illustration of the processes and their respective triggers which contribute to the exercise induced increase in skeletal muscle mass (click here for detailed elaborations).

If you take another look at the complex network of endo- and paracrine signalling cascades and the number of factors which contribute to a process that is generally reffered to as "skeletal muscle hypertrophy" (cf. figure 2) and is, at least in my mind, falsely reduced to the influx of amino acids into the muscle, it should be clear that testosterone does play a central role in the actual exercise induced growth response. That the latter is less pronounced than bro-science would have it (esp. when we are talking about physiological levels, cf. "Quantifying the Big T") and that testosterone itself and its metabolites, DHT and estrogen are probably of greater importance in the "restructuring" process, which in turn facilitate the accrual of even more protein within the muscle, does after all not imply that the huge differences in androgen levels are not the reason for the differential hypertrophy response in men and women - and I guess, I don't have to tell you that you just have to take a glimpse at the female IFBB (International Federation of Bodybuilding and Fitness) competitors to know that androgens can make a difference ;-)

Time Under Tension (TUT) Another Under-Appreciated Determinant of the Protein Synthetic Response to Exercise?

Image 1: Is it really time to buy some revolutionary new exercise equipment to time your time under tension? Or should you keep pumping away like there was no tomorrow?

If you have been following the SuppVersity news for some time now, you know that I am a "fan" of the research Stuart Phillips and his colleagues at the Department of Kinesiology at McMaster University in Hamilton, Ontario, are doing. Before I get to some details on their latest coup, I must yet express some concerns about Phillips' focus on immediate changes protein synthesis. Yes, amino acid ingestion and particularly leucine increase protein synthesis, yes, bolus ingestion of whey protein increases protein synthesis over sipping and yes, training with low loads (30%) and slow reps, as in the study at hand, increases protein synthesis,... but hey. Do you really give a damn about protein synthesis? No, you don't. Either you want to gain muscle or you want to get stronger and exactly here I am missing a link that would connect the short-term increases in the protein synthetic response to exercise Phillips and his colleagues are investigating in one study after the other and the long(er)-term real-world outcomes in terms of muscle size and strength gains.

Wait! Is protein synthesis really that important?

I will probably touch on this issue in tomorrow's installment of the Intermittent Thoughts, as well, so let me just say this: Muscle protein synthesis is only one out of two (maybe three processes) and probably not even the most important one, when your goals are getting really big or really strong. I mean, if increasing protein synthesis was all it would take to get as buffed as Phil Heath and as strong as Derek Poundstone, everyone would be training like a sissy (like in this study), take his BCAAs and whey protein and see amazing results... but I am once again getting off a tangent here, and as I already said, you will read more on that here at the SuppVersity in the future. So, for the time being, let's get back to the time under tension, i.e. the exact number of seconds your muscles are actually working (meaning contracting) during a given set.

Figure 1: Basic outline of the study first and second testing session (based on Burd. 2011)

For their most recent study Nicholas A. Burd et al. recruited 8 recreationally resistance-trained men (23.5 ± 1 years; 88.3 ± 5 kg; BMI=26.5 ± 1.0 kg/m²) who had performed lower body resistance exercise training with a frequency of at least 2x/week in the course of the last 2 years prior to the study [note: this certainly is a huge plus of the study, because we all know that you can have a newbie do nothing but climb stairs and he will still grow ;-] The individual one rep-max for leg-extensions was accessed once prior to the infusion trial (105kg right, 101kg left leg) and dietary intakes were recorded prior to both the resting and the exercise infusion trials, in the course of which the participants reported to the lab fasted (at 7am) before a catheter for the tracer infusion was inserted into their arm and a first (fasted) muscle biopsy was taken from their legs (3.5h after reporting to the lab).

Participants subsequently performed bouts of unilateral leg extension exercise at 30% of
their previously established concentric 1RM. Legs were randomized and balanced for dominance based on maximal strength to perform exercise at a slow lifting (SLOW) or an external work-matched control (CTL) conditions. The leg assigned to the SLOW condition performed exercise with a lifting/lowering cadence of 6 s concentric phase and a 6 s eccentric phase with no pauses until volitional fatigue (i.e. failure). Failure was defined as the point at which the participant could not lift through the full range or their technique to lift the load included motions at joints other than the knee. The CTL condition was completed with the contralateral leg and was matched to the experimental condition for contraction volume such that the leg performed an identical number of repetitions at an equivalent load, but not to failure, and was performed with a lifting cadence of 1 s concentric phase and a 1 s eccentric phase.

The participants performed a total of 3 sets with 2 minutes of rest between the sets for each condition. Lifting cadence was monitored by an instructor and by the use of a metronome. Moreover, the exact knee-joint angles were recorded by the means of a goniometer. After a subsequent 2nd blood sample was taken, all participants consumed 20g of whey protein isolate. 6h after, a 2nd bilateral biopsy was taken and the participants were fed a standard cafeteria meal. For the rest of the day they were advised to follow a diet that would mirror their previously recorded food intake, with the last meal being consumed before 22h, "to ensure a 10 h fast prior to the beginning of the 24 h post-exercise protein synthesis measurement", which took place the next morning after the consumption of another 20g of a tracer-enriched whey protein supplement.

Figure 2: Fractional protein synthesis (in % per hour) - left; and relative differences in protein synthesis of slow vs. ctrl condition - right (based on Burd. 2011)

The data in figure 2 clearly shows that going to failure (and this is what I consider even more important than time under tension when training with sissy 30%1RM loads) produces profound (compare the relative increases in the smaller graph on the upper right corner) increases in fractional protein synthesis, which are, in the time-window right after the exercise bout, particularly pronounced in the mitochondrial and sarcoplasmic compartment of the muscle. In this regard, Burd et al. point out that

[w]hat we observed here was a potentiated effect, from that seen in the fasted-state, of prior exercise in enhancing the feeding-induced myofibrillar protein synthetic rates. This effect appears to be dependent on maximal fibre activation during exercise, [...] The current study is noteworthy in that an enhanced effect of protein feeding during late exercise recovery was induced by a longer time under muscle tension rather than intensity-independent contraction volume, which we have previously examined (Burd. 2010).

As far as the delay in the normally immediate increase in myofibrillar protein synthesis is concerned, the researchers speculate that both the timing of the biopsies, as well as the training status of the subjects and the specificity of their protocol about which they state that with its long loading times at relatively low intensities it must have shifted the protein (immediate) myofibrillar protein synthetic response "toward increased synthesis of proteins in the mitochondrial and sarcoplasmic pools" (cf. figure 2) - a process the underlying causes and mechanisms of which are yet unclear.

Why would you change a winning team?

Image 2: When it comes to SST and all the other training types from the alphabet soup, I alway wonder why people keep questioning what has worked well for the majority of bodybuilders and athletes, they are looking up to and whose physiques they are admiring!?

Actually this observation takes us full circle to my introductory remarks on the possible short-sightedness of measuring acute fractional protein synthesis. After all, what we are seeing here is rather the response we would expect as a consequence to a rather endurance-oriented exercise regimen. Whether the latter would entail the "size" (and strength) gains everyone currently associates with the magic words "increases in protein synthesis" remains thusly highly questionable.

This is particularly true if we take into account the results of another pretty recent study be Eonho Kim et al. (Kim. 2011), which found that an even slower (10s concentric, 10s eccentric) training protocol at 50% or the 1RM led to greater increases in flexibility but highly variable and overall lower strength gains than a traditional protocol with (4s total TUT at 80%RM) in college-aged women. This basically confirms what previous studies by Keeler et al. (+39% in traditional, only +15% in slow training; Keeler. 2001) have already established: (Super) Slow Training works, but it does not work as well classic resistance training.

And no matter whether you train slow or fast - in the end, intensity will always be determined by a matrix of loads, volume, TUT and training density and I doubt we will see a study that controls for all this variables even in the remote future (and if that happens you know that the SuppVersity is the place where you will read about it, first) - so the best thing you can do, is to rely on what worked for generations of physical culturists and that was definitely not training with 6s concentric and 6s eccentric reps ;-)