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 ;-)

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.