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 ;-)

Whey or Casein? Which Would be the Better "Staple" Protein Source for Your Trip to Desert Island?

Image 1: They are both sourced from cow's milk, but which is the better part? Whey, the byproduct of cheese production, or casein the cheese protein, itself? A recent study would suggest that it's the "waste product" you would have to chose if you could only have one.

"Whey is the way to go!" I suppose even I have had a headline like that in one or even several of the daily news items, here at the SuppVersity - and rightly, so! With it's high content of branched-chain amino acids (BCAAs) this fast-digesting protein source is certainly the #1 choice for anyone whose goal is to build lean muscle tissue. Whey's slow-digesting brother casein, on the other hand, is often hailed as the "muscle-preservative", the 24h-protein source that will prevent muscle catabolism, when for whatever outrageous reason (like sleep, for example) you cannot ingest your bi-hourly protein shake... well, I guess those of you who have been following the Intermittent Thoughts on Intermittent Fasting will already be "rolling on the floor laughing", but hey! Do we really know whether casein or whey would be the better "staple" protein - I mean, if you sipped it throughout the day?

Casein vs. whey - which one to chose if you cannot have both?

While I would not say that one study could provide a definite answer to this question, the results of a recently published paper by Stéphane Walrand et al. (Walrand. 2011) provides further evidence that whey, not casein would be your best choice - regardless of the diminished return that comes with sipping it.

Figure 1: Ingredients of the 6 diets the rats in the Walrand study were fed for 5 months; CAS = casein, WHEY = whey (data adapted from Walrand. 2011)

In their long-term (5 months!) feeding study, the scientists supplied 21 week old male Winstar rats (at the beginning of the study the animals were thus "middle-aged") with one out of 6 experimental diets (cf. figure 1). The composition of the diets differed not only in their total energy and protein content (ad libitum = 440kj/day; energy restricted only 60%, i.e. 264kj/day), but also with regard to the protein content and source (casein vs. whey). In that, it is particularly noteworthy is that the "energy restricted" diet was actually a "high protein" diet. After all, the protein content of the latter was identical to the one of the rats that had free access to  (the group that was "only" energy restricted received was matched to the average protein consumption of the ad-libitum fed rats.

Figure 2: Effect of 5 months of the experimental diets on muscle and fat weight of male Wistar rats (data adapted from Walrand. 2011)

Contrary, to what you may have expected, the "protein deficient" protein & energy restricted diet did yet not lead to profound losses of lean muscle tissue (cf. figure 2). On the contrary, the protein & energy restricted group that received whey protein as their exclusive protein source had 5% and 2% greater soleus and tibialis anterior mass than the ones that received the "high protein" energy restricted diet. Before you start questioning the value of "high" protein intakes when dieting, you should yet better take a look at the impact of the "high" protein content of the non-protein-restricted diet had on the diet induced reductions of the abdominal fat mass. I mean -87% reduced abdominal fat in the energy & protein reduced group is impressive, the neigh complete annihilation of the abdominal fat (-93%) in the non-protein restricted group, on the other hand, is mind-boggling.

Figure 3: Effect of 5 months of the experimental diets on muscle and fat weight of male Wistar rats (data adapted from Walrand. 2011)

If we also consider the nitrogen balance and the absolute rates of muscle protein synthesis (cf. figure 3), it also becomes evident why the rats on the protein & energy reduced diets retained slightly more lean mass (+3%), when they were fed whey protein, instead of casein. The rats who received whey as their main protein source simply had a favorable nitrogen balance and increased muscle protein synthesis.

Image 2: Sardines for diabetes prevention!?

Before you now throw away your eggs, your cheese, your beef and whatever else, I want to briefly introduce you to the results of two other recently published studies, which would indicate that rotating in some sardines or sheep meat could produce even more favorable results than living on whey alone. While Madani et al. found that sardine protein ameliorated fructose-induced hyperglycemia, insulin resistance, hyperlipidemia and inflammation (vs. casein) in a 2-months rodent study (Madani. 2011), Feng et al. report that the consumption of sheep meat instead of casein lead to increases in free T3 (thyroid hormone) and statistically significant increases in energy expenditure in Sprague-Dawley rats that were fed otherwise identical diets (Feng. 2011).

Despite these and the results of previous studies, most of which would suggest that if you had to chose just one protein source, whey or casein, whey should be the protein of choice, I hope that I do not have to tell you, as a diligent student of the SuppVersity that imbalances are the root cause of many, if not most modern diseases. So, getting all your protein from whey and nothing but whey should not be something you should even remotely take into consideration. And in case you forgot about that: Milk has both of them and a ton of other vital nutrients ;-)

Green Tea for Muscle Protection? GTE Increases Satellite Cell Proliferation & Differentiation, Slows Disuse-Related Atrophy, Does not Promote Hypertrophy in Aged Rodents

Green tea as a magical muscle preservative for injured athletes?

"GTE increased satellite cell proliferation and differentiation, decreased oxidative stress and the abundance of Bax, a proapoptotic protein" (Alway. 2014) - that's the initially exciting result of a recent study from the West Virginia University School of Medicine and Abbott Laboratories. What is not exactly as exciting, though, is how the sentence continues, i.e. "yet this did not further improve muscle recovery in reloaded muscles" (Alway. 2014).

Sounds contradictory, right? Well, before we get deeper into the discussion of the results, let's briefly recap how Alway et al. arrived at these insights, i.e. how exactly the experiment looked like and which experimental evidence it generated.

The scientists from the West Virginia University School of Medicine tested the hypothesis that green tea extract (GTE) would improve muscle recovery after reloading following disuse. In men and women "muscle disuse" would equal lying around in bed or on the sofa all day. In rodents it was simulated by an initial 14-day period of hindlimb suspension (HLS) and a subsequent period of reloading (recovery).

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The subjects the researchers use were Fischer 344 Brown Norway rats who were randomly assigned to receive either 14 days of hindlimb suspension (HLS) or 14 days of HLS, followed by normal ambulatory function for 14 days (recovery). Additional animals served as cage controls.

Figure 1: Muscle wet weight. Muscle wet weight was obtained in hindlimb muscles of cage control animals, after 14 days of hindlimb suspension group (HLS), or after 14 days of hindlimb suspension followed by 14 days of reloading (Recovery). And Ex vivo isometric force. A. Maximal tetanic force obtained at a frequency of 100Hz, or B. Peak twitch force (PT) of the plantaris muscle was measured in cage control rats, after 14 days of hindlimb suspension (HLS) or after 14 days of hindlimb suspension followed by 14 days of reloading (Recovery | Alway. 2014).

Both active treatment groups were given green tea extracts at a dosage of 50 mg/kg body weight - that's roughly 600-750mg of green tea extract per day. The control group received pure water, instead.
As you can see in Figure 1, the animals that received the green tea supplement exhibited a significantly attenuated loss of hindlimb plantaris muscle mass and tetanic force during.

In addition, compared to the vehicle treatment, GTE attenuated muscle fiber cross sectional area loss in both plantaris (-39.9% vs. -23.9%, p<0.05) and soleus (-37.2% vs. -17.6%) after HLS. This green tea-induced difference was not transient but it was maintained over the reloading period.

Increased muscle retention = increased fat loss!?

That's particularly interesting in view of the fact that the changes in body weight did not differ between the green tea and water group (see Figure 2).

Figure 3: Relative reductions in total body body weight in the two groups (Alway. 2014)

Why? Well it signifies that there is a significantly reduced negative impact on the body composition, with green tea. That does not change, though, that "GTE failed to further improve recovery of muscle function or mass as compared to vehicle treatment" (Alway. 2014)

This begs the question - would you recommend GTE? As a muscle preserver during periods, where you cannot workout, yes. The way it conserved the muscle mass in the study at hand should help help you to get back to the grind after a debilitating exercise, even if the recovery in the study at hand seemed to be identical in both groups. The green tea induced increase in satellite cell proliferation and differentiation, as well as the decreased oxidative stress and the abundance of the catabolic protien Bax, on the other hand, is probably not going to have a significant effect as long as you are still able to move, because exercise alone will induce more pronounced benefits in these domains.

Figure 4: Changes in body composition in a study w/ obese subjects comparing GTE to resistance training and a combination of both green tea extract and resistance training on body comp. (Cardoso. 2013)

Plus: We should not forget that the effects were observed in old rats and thus in a model of a population group that appears to benefit from antioxidant supplementation more than young(er) people. If you don't belong to the corresponding group of human beings whose muscles are particular prone to oxidative damage and suffer from a reduced ability to adapt to exercise induced stress, the effects remain questionable. In obese individuals green tea has yet been shown to promote the beneficial effects of exercie on body composition (Cardoso. 2013) - the risk that it a low dose of GTE does anything but good, does thus appear to be very small; and that's true for the benefits for athletes, too - at least according to previously reported results | Comment on Facebook!

References:

• Alway et al. "Green tea extract attenuates muscle loss and improves muscle function during disuse, but fails to improve muscle recovery following unloading in aged rats." Journal of Applied Physiology (2014). Ahead of print.
• Cardoso, Gabrielle Aparecida, et al. "The effects of green tea consumption and resistance training on body composition and resting metabolic rate in overweight or obese women." Journal of medicinal food 16.2 (2013): 120-127.

HMB Exhibits Differential Effects on ATP and Glycogen Content of Fast & Slow Twitch Fibers and Maximizes Tetanic Force Development in Rodent Study

Image 1: This is where HMB could actually make a difference, the two more reps, the one more sprint, which after weeks and months of training can decide over victory or defeat.

Sometimes, or I should say, time and again (!), it amazes me how the same people who are willing to invest hundreds of bucks in a supplement, which (according to the patent holder) "has been shown in scientitfic studies" (which were conducted by the researcher and a buddy of his at a remote lab, only to file the patent) to "increase testosterone by up to 147.34%", keep telling me that they "would never waste their hard earned money on supplements like HMB..." hello? Am I missing something, here? I mean, right; HMB does not produce the steroid-like effects the same sort of shady businessmen who are now promoting a new natural testosterone booster as legal alternative to Anavar on a monthly base once claimed it would have, but in all  honesty, the scientific research on HMB is by far more promising than the mostly non-existent research on 99% of the "legal anabolics" out there.

HMB works, we just don't know exactly how and for whom

As Dr. Connelly pointed out on the last BodyRX Show (highly recommended, especially for Layne's intellectual exchange with Dr. Jeff Volek), it stands out of question that HMB works. There are in fact more than a dozen of studies which show that supplementation with adequate amounts of this leucine metabolite has anti-catabolic effects in various conditions of skeletal muscle atrophy (Nissen. 2003; Smith. 2005). What  is still debatable, though is whether and to which extent athletes, in general, and bodybuilders, in particular can benefit from these effects. In view of the increasing awareness of the importance of leucine, the metabolic precursor of b-hydroxy-b-methylbutyrate (HMB), most athletes in this subgroup probably consume somwhere between 20-30g of leucine from the 300g of protein they are feeding themselves in the form of protein shakes and lean meats alone (with reference to the data that is presented in figure 1 it is noteworthy that the comparison Nissen made is not "fair", because the many of the HMB studies were conducted with "sick" people, while the majority of studies on protein supplements used either healthy people or athletes). With an average conversion rate of ~5% (of dietary leucine), we would have to estimate their daily HMB "production" to roughly 1.0-1.5g, which is interestingly at the lower range of what has been shown to ameliorate muscle wasting in cancer cachexia studies (Eley. 2007; Kovarik. 2010).

Figure 1: Calculated effect sizes of creatine, HMB, chromium, androstendione, DHEA and protein supplements on strength and lean mass gains (adapted from meta-review by Nissen et al.; Nissen. 2003)

And even if we discard the question of whether or not additional HMB is really necessary on a high protein diet and whether or not respective dietary differences could explain the negative results from some, yet by no means all, trials with professional athletes, we must still admit that even in those cases where it does work, we (i.e. scientists) do not really understand how HMB does its anti-proteolytic magic. The results of a recently published study from scientists from the Institute of Biomedical Science at the University of Sao Paulo, could thusly be of particular importance, as this is - at least to my mind - the first study to investigate the effects HMB supplementation had on ATP and glycogen levels, citrate synthase and changes in the contractive properties of individual muscle fibers (Pinheiro. 2011).

Figure 2: Changes (vs. placebo) in ATP and glycogen content, as well as citrate synthesis in red and white portion of rat gastrocnemius muscle after 4 week supplementation with 320mg/kg HMB (data adapted from Pinheiro. 2011)

The data in figure 2 shows, that after 4 weeks of daily supplementation with 320mg/kg HMB (in the study the usual calcium salt, you can buy in bulk on the Internet was used), the ATP and glycogen levels in the gastrocnemius muscle of the rats were profoundly elevated. In that, it is particularly interesting that the leucine metabolite had differential effects on the slow-twitch oxidative red portion of the muscle and the fast-twitch glycolytic white portion: In the slow twitch fibers the increase in ATP is 10x higher than it is in the fast twitch fibers, where the +400% increase in glycogen content should yet provide a similarly extensive buffer of readily (yet not immediately) available energy. Moreover, the increase in citrate synthesis (+67%) in the slow twitch fibers suggests that part of this effect was mediated by an "increased lipid availability due to increased lipolysis", or, put simply, by an increased oxidation of fatty acids to generate more ATP.

Figure 3: Tetanic force production (normalized to muscle weight) in rats receiving 320mg/kg HMB or placebo for 4-weeks; successive tetanic contractions were evoked at 100 Hz each 10 s of interval (data adapted from Pinheiro. 2011)

These increase in both readily available energy stores and the ability to replenish the former via fatty acid oxidation, is - according to Pinheiro et al. - also the underlying reason for the "increase in resistance to fatique" the scientists observed when they subjected the rat muscle to electrical stimulation in order to evaluate the tetanic (=constantly contracting) force production (cf. figure 3). Contrary to the twitch force, which was identical in supplemented and non-supplemented rats, the tetanic force production (normalized for either muscle weight or muscle cross-sectional area) increased by +17% (p<0.05; meaning that the chance that this was mere coincidence is <5%).

Fine!? Now, tell me: Is HMB worth it?

In view of the fact that neither the muscle size (cross-sectional area) nor the lean mass of the rodents in the HMB group differed from their placebo supplemented peers (btw. the animals were not "trained" in the course of the 4-week study), we must conclude that the effects of HMB, similar to those of creatine, are not what you would call "immediately anabolic". In a real world training scenario the metabolic advantage (increased ATP stores, increased glycogen stores and increased oxidative capacity) the rats in the HMB group gained over the 4-week study period, would allow trainees to do those 1-2 reps more which in weeks and months would then translate into this one additional pound of muscle or the 10th of a second that can make the difference between victory or defeat - whether those 1-2 reps are worth the roughly 64$ it would cost to copy the supplementation regimen used in the study (320mg/kg in rats would equate to 53mg/kg per day for humans), does yet depend on who you are, what you want to achieve and how much money you have to spend... and if you do not have your regular diet and training in check, don't even think of HMB (let alone one of those "test boosters" ;-)

Citrulline = The Dieter's Amino Acid? Citrulline Maintains Muscle Protein Synthesis & Strength Endurance During Caloric Deficits Better Than Leucine!?

Can citrulline supplementation prevent you from hitting a catabolic wall, when you are dieting? And is it more potent than leucine?

You have been told "leucine is the most anabolic amino acid known to man", by the guy at your local GNC, the bros in the gym and the "experts" on the board.

And yeah, in a way, they all are "right", but the surprising negative effects of HMB supplementation on the muscle catabolism during overtraining (read more) should have reminded you that this does not imply that it will also protect your muscles against muscle breakdown and/or have similar "anabolic" effects on a diet.

Dieting is a major change in the metabolic stage and another stage means a different cast, among whom citrulline could turn out to be the new star... at least if we trust the results of a recent rodent study.

Different metabolic stage - new stars on the scene

In their most recent paper Ventura et al. describe the results of a rodents experiment in the course of which they  evaluated the effect of sequential administration of leucine (LEU) and citrulline (CIT) to preserve lean body mass during food restriction. In a 2009 study, Moinard et al. had already observed that the provision of 1.0 g/kg/day of CIT (HED ~10-15g) to exert beneficial effects on body composition in aged rats (Moinard.2009) and if you go by the abstract of the study at hand, it would sound as if citrulline was not simply "lean mass protective", but also much more potent than leucine:

Only CIT administration (1 g/kg) was able to restore MPS [muscular protein synthesis] (CIT1: 3.4±0.3 vs.R: 2.5 ±0.2 %/day,p=0.05) and increase muscle maximum tetanic force (CIT1: 441 ±15 vs.R: 392 ±22 g,p=0.05) and muscle strength (CIT1: 4,259±478 vs. R: 3,045 ±663 A.U., p=0.05). LEU had no effect and CIT+LEU supplementation had few effects, limited to adipose mass and fatigue force. The results of this study highlight the ability of CIT alone to preserve muscle function during dietary restriction. Surprisingly, LEU antagonized some effects of CIT." (Ventura. 2013)

This observations have been made after the rats dietary provisions had been cut by 60% for 2 weeks while the amino acid composition of their diet had been increased by the provision of additional amino acids: 

• R-CIT 0.2 - low dose citrulline: 0.2g/kg
• R-CIT 1- high does citrulline: 1.0g/kg
• R-LEU - leucine: 1.0g/kg
• R-LEU-CIT - leucine + citrulline: 1.0g/kg + 1.0g/kg

By addding valine (130 mg/kg/day) and isoleucine (220 mg/kg/day) to the diet, the researchers had also ensured that the natural BCAA balance would be maintained and ....

Figure 1: Changes in body composition during the 2 weeks on 60% of the regular energy intake with different amino acid supplements in the diet (Ventura. 2013)

... well if you look at the "net result" in terms of weight loss, it would in fact seem that citrulline is the way to go... if you do yet take a look at the lean mass measurements, it becomes plain obvious that there was no difference to the starved control group in any of the AA supplemented rodents.

Figure 2: Muscle contractile properties (fatigue AUC), myofibrillar and sarcoplasmic protein synthesis (PS) after 2 weeks on the different 40% dietary restricted diets (Ventura. 2013)

This is interesting, as it stands in contrast with the directly measured influx of protein into the myofibrillar part of the skeletal muscle of the rodents, and does not mirror the pronounced benefits on muscular fatigue the researchers observed and is not appropriately discussed in the study, the authors of which were so fascinated by the miniscule increase in protein synthesis that they did not even notice that they effectively produced a null-result.

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The hormonal response (esp. testosterone & GH) to workouts is another of those things that don't predict real world results (learn more)

Real results count: So does it really matter that the protein synthesis increased? No, just as it does not matter in the countless post-exercise protein synthesis studies. If you want to inflate a tire, you are not interested in how much air you can pump into it, but rather how much of the air will stay inside and the results of the study at hand only confirm that the former cannot predict the latter.

And let's face it: None of the treatments actually had to prevent lean mass loss, because much contrary to the bro-scientific believe that you would lose tons of muscle mass within a day, if you don't get all your shakes and pills in just in time. The rodents lost no lean mass at all.

So if you want take home messages, don't rely on protein synthesis rates alone and don't freak out about muscle loss too much.

True or False: α-Hydroxy-Isocaproic Acid aka HICA is a Potent Anti-Catabolic, Just Like the Shiny Ads Say

Even Arnold benefited from α-hydroxy-isocaproic acid aka HICA - the HICA his body produced and the HICA he got from his diet, whenever he ate cheese and other fermented foods.

You know that I am not the kind of person who likes to tell others what to do. After reading my summary of the contemporary research on α-hydroxy-isocaproic acid aka HICA, you should yet be able to decide whether it's worth a try or not.

If you take a look at the pertinent databases you will realize that there are more patents than papers on α-hydroxy-isocaproic acid - usually, this is a good indicator we are dealing with another industry scam, but in contrast to the many funky forms of creatine, α-hydroxy-isocaproic acid does actually have a handful of studies to back up that it does... or I should say "that it could" help you getting big and buffed.

If I had to chose between HMB and HICA, I would choose HMB... or better stick to whey!

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That being said, it may be worth taking a look at what α-hydroxy-isocaproic acid actually is. Just like HMB which is about to make a comeback in liquid form, these days, HICA is a metabolite of the mTOR and thus protein synthesis triggering branch-chained amino acid leucine. It is also known as "leucic acid" or "DL-2-hydroxy-4-methylvaleric acid" and is formed by α-hydroxylaction from leucine. It's one of the end-products of leucine metabolism in muscle and connective tissue (Walser. 1978). It's usual concentration in our blood is about 0.2560.02 mmol/L - that's ~100x less than the amount of α-keto-isocaproic acid (KIC), the corresponding keto acid of leucine of which you'll find more than 21 mmol per liter in your blood.

Figure 1: Relative changes in lean mass (%) during 4 weeks of intense soccer training on 1.5g/day HICA (Mero. 2010)

Cheese, wine, soy sauce, etc. - the recently celebrated fermented foods, they all contain HICA, which appears to be the anti-catabolic counterpart to leucine. While the latter is a potent promoter of protein synthesis, the former appears to make sure that the work of its predecessor leucine is not lost.

It is thus no wonder that the promising results of a 2010 study by Mero et al. were recorded during an intensive and thus potentially catabolic training period in soccer athletes. In contrast to the placebo group, where only one individual gained a significant amount of lean mass, while 4 lost muscle, the subjects who had been consuming 1.5g/day of α-hydroxy-isocaproic acid gained 300g of lean mass, on average, in the course of the 4-week study.

That's not much and it was not fat free (ca. 150g of fat), but the data in Figure 1 shows that this is a difference between minimal muscle loss and gain... and I guess most athletes would prefer a marginal muscle gain over a marginal loss of lean mass.

HICA, a potent anti-catabolic? I don't think so!

The notion that HICA is, above all, a muscle loss inhibitor appears questionable, if we take a look at the results Charles H. Lang, Hugues Magne, Elizabeth Offord and Denis Breuille presented at a 2013 FASEB meeting. In the abstract to their presentation they cite the results of a rodent study in the course of which the rodents were immobilized for two full weeks. The consequence, an increase in the expression of catabolic hormones and a profound loss of muscle mass was identical in both the HICA and placebo supplemented groups, but in spite of the fact that "αHICA did not alter the immobilization-induced increase in proteasome activity and atrogene expression", the muscle mass had returned to control values only in αHICA-fed rats after 14 days.

No performance enhancing effects: In spite of the increase in muscle size (or should I say absence of a decrease?) Mero et al. didn't record any performance enhancing effects of HICA in their study w/ professional soccer players. Minimal muscle gain, yes. Reduced DOMS, yes, even that. Increased performance? No. As the authors point out, the study period (4 weeks) may have been to short. That's correct, but if you take another look at the data in Figure 1 you would still expect to see marginal differences, at least, right?

The fast recovery in the HICA group was associated with increased muscle protein synthesis and higher levels of the "protein synthesis pump initiator proteins" S6K1 and 4EBP1 in the previously immobilized muscle. The results Lang et. al. have not yet published in a full paper (at least I couldn't find it) put a huge questionmark behind the long-heralded hypothesis that HICA supplementation would slow muscle loss and puts it in line with its cousin HMB and its precursor leucine as a purported pro-anabolic muscle builder.

Figure 2: Gastrocnemius weight (rel. to control) immediately before and 14-days after the immobilization (Lang. 2013).

The data from the corresponding full paper the authors published a couple of month later in the American Journal of Physiology - Endocrinology and Metabolism you see in Figure 2 are even more impressive, though. According to this rodent data HICA is a more potent muscle (re-)builder than leucine; and, importantly, neither of the two does what the industry keeps promising: prevent muscle catabolism in response to disuse.

Bottom line: A confirmation of Lang's results in humans and/or a resistance training scenario like the one Wilson et al. did for the free acid form of HMB recently ("Breakthrough HMB Research: Additional(!) 10% Reduction in Body Fat, 5% Higher Lean Mass + 2x Higher Strength Gains After 12W of Heavy Lifting in Trained Individuals" | read more) are yet still missing. Aside from the previously cited soccer player study by Mero et al. we do have...

• a paper by Chow & Walser (1975) who report that leucine and its α-hydroxy analog (HICA) promote muscle growth equally effective, although replacement of leucine with HICA reduced food intake and increased the volume of urine and its nitrogen concentration
• a study by Woods & Goldman (1979) who report that HICA can be used as a leucine replacement in the diet without reducing food intake or growth of the animals

...and thus not enough arguments for me to spend money on currently hilariously overpriced α-hydroxy-isocaproic acid, but I am running a non-profit blog, so if you are making big money with a website or whatever else and want to give it a try - there is no evidence that HICA may harm anything but your purse.

Reference:

• Chow K., and Walser M. "Effects of substitution of methionine, leucine, phenylalanine, or valine by their alpha-hydroxy analogs in the diet of rats." J Nutr 1975;105(3):372 8
• Lang, Charles H., et al. "Chronic α-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy." American Journal of Physiology-Endocrinology and Metabolism 305.3 (2013): E416-E428.
• Mero, Antti A., et al. "Effects of alfa-hydroxy-isocaproic acid on body composition, DOMS and performance in athletes." Journal of the International Society of Sports Nutrition 7.1 (2010): 1.
• Walser, Mackenzie. "Therapeutic compositions comprising alpha-hydroxy analogs of essential amino acids and their administration to humans for promotion of protein synthesis and suppression of urea formation." U.S. Patent No. 4,100,160. 11 Jul. 1978.
• Woods M., and Goldman P. "Replacement of L-Phenylalanine and Leucine by a-Hydroxy analogues in the diets of germ-free rats." J Nutr 1979;709:738 43.

Where Protein Fails, Protein + Resistance Training Succeed: Lifting Corrects Diet-Induced Decrease in Postprandial Protein Synthesis, But Fails to Normalize Net Retention

It takes pains to maintain your gains!

You will certainly remember the shocking revelation that simply eating more protein is not going to prevent the diet induced muscle loss that occurs whenever you consume less energy than you expend (read up on "Protein Intake & Muscle Catabolism: Fasting Gnaws on Your Muscle Tissue and Abundance Causes Wastefulness" | go for it!)...

Don't rejoice, the study at hand does not refute this - protein is still unable to counter the increase in atrogin-1 and other muscle cannibalizing proteins, but there is a "tweak" by the means of which you can at least avoid that its pro-anabolic affects are also impaired.

You can learn more about protein intake at the SuppVersity

Are You Protein Wheysting?

Cod protein for recovery

Protein requ. of athletes

High EAA protein for fat loss

Fast vs. slow protein

Too much ado about protein?

What this "tweak" is? Well, that's easy: Heavy lifting. If you are familiar with the "muscle loss in zero gravity" research that has been conducted by and for the NASA in the past decades (e.g. Ferrando. 2002).this shouldn't surprise you. The NASA studies have after all shown quite conclusively that compared to bed-rest / chronic skeletal muscle unloading, starving yourself is almost "anabolic". No wonder that lifting heavy objects, and not dietary protein is the #1 when it comes to saving your muscular ass from shriveling away on a long and hard diet.

Why does resistance training work, if protein fails?

As discussed in "Protein Intake & Muscle Catabolism" (read it!), it's not a question of the pro-anabolic effects. You, as a suppversity reader know that the p-AKT/mTOR pathway that's activated by protein feeding is sufficient to increase the influx of protein into the musculature. What your beloved protein can't do, though, is to reset a different switch: The "sacrifice muscle to fuel more fundamental metabolic demands switch" which is triggered whenever you are in a long(er) term energy deficit.

"Training For Gains: High Intensity, Low Volume Strength Gains Stick." | more

So what can be done then? Well,... as it is so often the case, the answer lies - once more - open before our eyes: Hit the weights, down the protein and kick your diet's catabolic ass!

I know this sounds too easy, but if you take a peek at the weight loss diets of the average physique athlete and their appearance on stage, it stands out of question that the combination of resistance training and strategic protein supplementation spares muscle mass.

Now the verb "to spare", according to the Oxford English Dictionary, means "to leave (a person) unhurt" (OED.COM), which is - and you probably expected this already, not really accurate. Even the latest data from the School of Medical Sciences at the RMIT University in Melbourne and the Exercise Metabolism Research Group at the Department of Kinesiology of the McMaster University in Hamilton, Ontario, Canada, and the Canadian Sport Institute clearly demonstrates that you cannot switch the diet-induced protein wasting off, completely (Areta. 2014).

Figure 1: The large inter-individual differences make it virtually impossible to tell, whether the MURF-1 levels increased. The similarly catabolic (see overview in the middle) atrogin was yet significantly increased in the early (15g) and late phase (30g) after the workout during ED (Gumucio. 2013; Areta. 2014)

In the corresponding experiment, 16 young, healthy, resistance trained subjects (8 females, 8 males) who had been fed individualized pre-packaged meals delivering 45 kcal/kg FFM (macros: PRO / CHO / FAT 1.4-1.6, 3-3.5 and 0.5-1.5 g·kg BM) per day for five days before they went on a standardized energy 30% energy reduced diet containing approximately

• 1.4-1.6g protein per kg total body mass, 
• 4.0-4.5g carbohydrates per kg total body mass and
• 1.5-2.5g fat per kg total body mass

for another five days. At the end of this "ED" period and five days on rations with only 30kcal/kg fat free mass, all subjects performed a standardized leg press workout (warmup + 6 sets of 8 repetitions at ~80% 1 RM with 3 min rest between set) that was followed by the ingestion of either 15g or 30g of whey protein or an isocaloric placebo.

Figure 2: SLC7A5 AA transporter expression (left) and myofibrillar fractional protein synthesis (% / hour; Areta. 2014)

What a brief glance at the data in Figure 2 does tell you, though, is that resistance training will effectively counter, the diet-induced downregulation of the pro-anabolic response to protein. What it won't do, though is to increase the net protein retention to levels comparable to those on an energy balanced diet!

A high protein intake doesn't normalize the levels of anabolic hormones, either | learn more

Loss ￪, synthesis down ￬ ➲ net protein loss - there is no way out! In conjunction with the concomitant reduction in protein synthesis (-27% in the study at hand), the combination of increased loss and decreased synthesis in a caloric deficit will always entail a net loss of protein (also in view of the endocrine deterioration | learn more). What exercise can do for you, though, is to counter the net-reduction in protein synthesis, i.e. maximize the amount of amino acids that is pumped into the muscle, before it's used for hepatic gluconeogenesis.

Contrary to what Areta et al. may have suspected the restoration of the protein synthetic response in the post-workout period did not restore the expression of the amino acid transporter gene SLC7A5 to normal. It is thus not surprising that...

Highly suggested read: " Evidence From the Metabolic Ward: 1.6-2.4g/kg Protein Turn Short Term Weight Loss Intervention into a Fat Loss Diet" | more

"[...] despite this elevation, exercise merely restored MPS [muscle protein synthesis] to a level that was similar to, but not exceeding, rates measured in EB [energy balance]. Accordingly, it appears the metabolic status of the muscle during short-term (5 days) ED [energy deficit] plus a ~10 h fast may dictate that contractile overload in isolation is not enough to increase MPS to values that otherwise would be observed when subjects are in EB." (Areta. 2014)

The results of this recent study do thus have to regarded as another nail an already boarded up coffin that's loaded with bro-scientific myths about "body recompositioning."

A word on "body recomposition": You cannot build muscle, while you are dieting. You can, however improve your body composition by losing more fat than muscle. In the mirror / on photos, the results will look like "gains" - in spite of the fact that you simply revealed the muscle that has always been hidden beneath the blubber.

Unlike the non-existent changes in amino acid transporter expression, the observation that 30g of protein are more effective than 15g will probably not come as a surprise to you - notwithstanding the fac t that this was "the first [study] to determine the acute muscle anabolic response to resistance exercise with two different doses of protein ingested after exercise during short-term ED", by the way. About as unsurprising as the researchers' (eventually unwarranted - I don't see a 20g protein group, here ;-) conclusion that their ...

"[...]results suggest that the optimal amount of protein to maximize the response to a single bout of resistance training while in ED may be above the level (20 g) found to maximize MPS post-exercise for individuals who are in EB." (Areta. 2014)

And my recommendation, not to worry too much about all the details. There are a couple of simple principles that have been working for generations of athletes thriving to cut weight without having to sacrifice muscle mass; and as you should know if you've read and memorized the "9 Simple Rules Every Dieter Must Follow" (go back) consuming 30g of protein with every meal and lifting heavy objects are both part of a set of rules that's rooted in bro- and supported by pro-science.

"There is Such a Thing As Over- training, Beware! When IGF-1 & Co Plummet and MAFbx Gnaws Away Your Muscles, It'll Be Too Late to Acknowledge" | more

Bottom line: In the end, the results of this study are probably less exciting than the title, i.e. "Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit" may have suggested.

That's yet not the least owed to the fact that you all know what it takes to maximize lean mass retention. If there wasn't that irrational hope somewhere deep inside your head that there was a hitherto unknown non-pharmacological way to build muscle and lose body fat at the same time, you'd now be hitting the weights or enjoying your post-workout protein shake... ;-)

Reference: 

• Areta, José L., et al. "Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit." American journal of physiology. Endocrinology and metabolism (2014). Ahead of Print.
• Ferrando, Arny A., Doug Paddon-Jones, and Robert R. Wolfe. "Alterations in protein metabolism during space flight and inactivity." Nutrition 18.10 (2002): 837-841.
• Gumucio, Jonathan P., and Christopher L. Mendias. "Atrogin-1, MuRF-1, and sarcopenia." Endocrine 43.1 (2013): 12-21.