Synephrine More Ergogenic, Than Thermogenic? Pump Supps Revisited - L-Arginine, L-Citrulline and Respective Whey-Peptides & -Nitrates | ISSN Research Review '15 #2

Synephrine, arginine, citrulline - Which pre-workout ensures that you're "on fire"?

As I pointed out previously, my initial idea to cherry pick only the most interesting study results that were presented in form of of poster presentations at the Twelfth International Society of Sports Nutrition (ISSN) Conference and Expo in 2015 didn't work out. The number of interesting studies is is simply too much for a single SuppVersity article to discuss them all.

Accordingly, I am now posting the 2nd serving of what is going to be a multi-part series of articles with brief discussions of the most significant results of the >20 studies and short references to those that didn't make the SuppVersity cut in this, previous and future installments of this series for one reason or another.

Read more about ISSN and other studies at the SuppVersity

Vitargo, Red Bull, Creatine & More | ISSN'15 #1

Pump Supps & Synephrine & X | ISSN'15 #2

High Protein, Body Comp & X | ISSN'15 #3

Keto Diet Re- search Update | ISSN'15 #4

The Misquantified Self & More | ISSN'15 #5

BCAA, Cholos-trum, Probiotics & Co | ISSN'15 #6

• Synephrine More Ergogenic Than Thermogenic? There was not just one, but two poster presentations and a full paper that has been published only days ago on synephrine containing supplements at the ISSN meeting and on the ISSN website, respectively. Synephrine? Yes, that's the supplemental non-starter, ah... I mean allegedly powerful fat burner from orange peels. The one with promising fat loss results in rodents, but discouraging results in practice.
  
  Luckily, the studies that were presented in form of posters by scientists from the Texas A&M University (Jung. 2015 & Dalton. 2015) at the ISSN meeting did not deal with synephrine as fat burner. Rather than that, Jung et al. and Dalton et al. took a look at the short- and long-term safety of synephrine as a pre-workout. A pre-workout that contained either 3g beta alanine, 2g creatine nitrate, 2g arginine AKG, 300mg N-acetyl tyrosine, 270mg caffeine, and 15mg Mucuna pruriens, alone (PLA) or the same baseline ingredients and synephrine.
  

  

  Figure 1: Number of reps on sets 1-3 & 4-6 in the control and treatment conditions (Ratamess. 2015)

  Now, the fact that some synephrine in your preworkout won't kill you is not really exciting. I have to admit that. What is exciting... at least sort of, though is the fact that the questionable thermogenic turned out to be an effective ergogenic in the already published and thematically related study by Ratamess and colleagues (Ratamess. 2015). A study that shows that p-synephrine of which previous studies indicate that it is a potent, but highly selective β-3 adrenoreceptor may nor be the best fat burner (the good old ephedrine was a pan-receptor activator and clenbuterol & co target the β-3 receptor, maybe that's also why the fat loss results are rather disappointing) , but at least an underestimated ergogenic.

Is p-synephrine different from synephrine? That's a good question without a clear question. Most supplements that list synephrine on the label actually contain P-hydroxy-α-{methylaminomethyl}-benzylalcohol aka p-synephrine, a protoalkaloid compound that differs from m-synephrine and o-synephrine structurally and comes in form of to stereoisomers in most supplements - the l-enantiomer and the d-enantiomer as the racemate d,l-synephrine. While the latter have been shown to be present in bitter orange, other forms, like the m,s-isomer may are suspected to be adulterations from synthetic phenylephrine supplement producers use to "spike" (Allison. 2005) their products (I assume this was not the case with the samples the researchers in the study at hand used, but the chaos wrt to the types of synephrine puts a huge "?" behind the assumption that you'll see the same effects from any given synephrine or synephrine + caffeine supplement. 

• As the data in Figure 1 goes to show you, the p-synephrine supplement, which was administered to twelve healthy, college-aged men at a dosage of 100mg either alone (S) or in conjunction with 100 mg caffeine (SCF) for three days. On the day on which the subjects participated in a standardized resistance exercise protocol consisting of 6 sets of squats for up to 10 repetitions per set using 80 % of their one repetition-maximum (1RM) with 2 min of rest in between sets, the supplement was ingested 45 minutes before the workout. In comparison to the placebo treatment synephrine alone triggered a significant increase in total repetitions and volume load. When synephrine was combined with 200 mg of caffeine, it also increased the mean power and velocity of squat performance. What did not change in response to either synephrine alone or caffeine and synephrine, though, were the blood lactate levels or the rate of perceived (RPE) exertion the subjects reported on the usual visual analogue scales.
  
  For me personally, that's a surprising result. For the scientists it "indicate[s that] supplementation with S and SCF can enhance local muscle endurance during resistance exercise" (Ratamess. 2015); and I have to admit: They are right. One thing you should keep in mind, though, is that unlike caffeine, where you often see reductions in RPE and thus an effect you will feel, synephrine will - even if it works - do its purported "magic" more subtly.
• The Latest on "Pump Supplements" - Creatine, Arginine, Citrulline, Nitrates -- To make sure that this series is not turning into a 12-part article, I will address the results of Moon's, Suzuki's and Vogel's results in one item (since Moon's paper basically summarizes the results of studies by Falcone and Joy, I won't discuss those separately) .
  
  

  

  Figure 2: Increases in blood flow volume (BFV) 33 minutes after the ingestion of 1.87g of RC, 3.67g of CP (citrulline content 1.87g), 1.87g of RA, or 3.07g of AP (arginine content 1.87g) before 3 sets of 15 arm curls (Moon. 2015).

  Moon et al.'s comparison of citrulline's and arginine's ability to increase the exercise-induced vasodilation and blood flow yielded unsurprising results that confirm that citrulline-based ingredients are more effective than arginine-based ingredients for modulating vasodilation and blood flow. Now that alone wouldn't be news-worthy if the scientists had not tested the effects of both, rawe L-citrulline (RC) and raw L-arginine (RA) and, citrulline and arginine bound to a whey peptide (CP and AP, respectively).
  
  I guess I am not going to surprise you, when I tell you that regular arginine was the worst vasodilator in this quartet. What is more surprising, though, is the extent to which the peptide bonding increased the vasodilating effects of regular citrulline and even arginine. With the the former producing 2x higher increases in vessel diameter and 9% higher increases in blood glow volume than regular citrulline (let's not even mention regular arginine | see Figure 2) the effects are pronounced enough to be potentially "feelable" and "visible" during a workout.
  
  

  

  Citrulline & Glutathione - GSH Amplifies & Prolongs CIT's NO Boosting Effects During + After Biceps Workout | learn more.

  Against that background I would be curious to see, whether the likewise recently reported performance enhancing effects of citrulline Suzuki et al.'s observed in twenty two well-trained young men who consumed 2.4 g / day of L-citrulline or placebo for 7 days and they took 2.4 g of L-citrulline or placebo 1 hour before 4 km cycling time trial on day 8 would be enhanced by bonding citrulline to whey peptides.
  
  I mean, if citrulline-whey-peptides appear to offer the same effects citrulline does, but at a higher efficacy, their beneficial effect on cycling time trial performance of which the Suzuki et al. argue that it was mediated by an up-regulation of plasma NO availability should be superior to that of raw citrulline, as well, right?
  
  And now that we are already talking about what really matters, i.e. performance enhancements, not increases in blood flow, it would also be interesting to see a follow up on the last "pump supplement" study to be mentioned in this installment of the ISSN Research Review: A study by Vogel et al.'s on the vasolidating effects of an arginine-nitrate based pre-workout supplements that shows significant increases in brachial artery diameter. Increases that are quantitatively similar to those Moon et al. observed for citrulline-whey-peptides in the previously discussed study. That does not mean, though that they are equally effective, though. To be able to say that we would need a study in which both agents are compared head-to-head. Unless this study is done and a follow up on Vogel's study proves that the increases in blood flow will actually produce significant performance gains, the only thing I can say about arginine nitrate is: It's interesting, but with respect to its ergogenic effects more studies have to be done.

You don't believe citrulline can do anything for you? Check this out: 8g/day Citrulline Increase Leg Workout Performance - More Reps on Leg Press, Hack Squat & Leg Ext. in Exp. Gymrats (more)! It is thus by no means useful for "cosmetic pumps", only 

Studies that didn't make the cut in this issue are Brooke Bouza et al.'s study on the exercise and calorie information on menus (Bouza. 2015) as well as O'Conner et al.'s tart-cherry study (O'Connor. 2015). That's not because there were methodological issues or something. It's much simpler: The notion that "exercise and calorie information on menus is not enough to improve food choices in Hispanic adults" (Bouza. 2015) is about as unsurprising as the word "potentially" (O'Connor. 2015) in the conclusion of O'Connor's study is daunting. And by the way, now that you know that tart cherry "potentially increases running performance and attenuates post-race markers of inflammation" you actually know the most relevant finding of O'Connor's study, right? | Comment on Facebook!

References:

• Allison, D. B., et al. "Exactly which synephrine alkaloids does Citrus aurantium (bitter orange) contain?." International journal of obesity 29.4 (2005): 443-446.
• Bouza, Brooke, et al. "Exercise and calorie information on menus is not enough to improve food choices in Hispanic adults." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P3.
• Dalton, R., et al. "Safety and efficacy of a pre-wrkout dietary supplement with and without synephrine." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P5.
• Falcone, Paul H., et al. "Acute hemodynamic effects of L-arginine, arginine nitrate, and arginine peptide on exercise-induced vasodilation and blood flow in healthy men." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P10.
• Joy, Jordan M., et al. "A comparison of raw citrulline and citrulline peptide for increasing exercise-induced vasodilation and blood flow." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P18.
• Jung, Y. P., et al. "Effects of 8 weeks pre-workout dietary supplement ingestion with and without synephrine on blood chemistry panel." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P4.
• Moon, Jordan R., et al. "A comparison of citrulline and arginine for increasing exercise-induced vasolidation and blood flow." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P6.
• O'Connor, A., et al. "Short-term powdered tart cherry supplementation encircling an acute endurance challenge potentially increases running performance and attenuates post-race markers of inflammation." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P7.
• Suzuki, Takashi, et al. "Oral L-citrulline supplementation enhances cycling time trial performance in healthy well-trained males." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P52.
• Vogel, Roxanne M., et al. "Acute hemodynamic effects of a multi-ingredient performance supplement on brachial artery vasodilation and blood flow volume following elbow flexion exercise in healthy young men." Journal of the International Society of Sports Nutrition 12.Suppl 1 (2015): P28.

Are You Afraid that the Fructose Boogieman Clogs Up Your Liver? Citrulline or Alanine, Glycine, Proline, Histidine and Aspartate Mix Will Protect You + Maybe Lean You Out

If you belong to the people who simply cannot stay away from HFCS foods and beverages, you may be happy to hear that the equivalent of as little as 10g citrulline or NEAAs in your diet may do much more than "just" fully prevent its negative effects on your liver.

You will probably remember from previous articles I wrote that NAFLD, or rather the development of non-alcoholic fatty liver disease, is one of the earliest markers of metabolic syndrome and beginning type II diabetes. In the Western obesity societies in North America and Europe, NAFLD is among the most common causes of chronic liver disease and its prevalence is increasing rampantly (Marchesini. 2001).

In spite of the fact that its development is most strongly linked to the consumption of a generally unhealthy, energetically dense diet, there are several lines of evidence which suggest that the ingestion of exorbitant amounts of fast-digesting fructose from high fructose corn syrup (HFCS) sweetened beverages or processed foods is one, if not the most reliable motor of its development (Volynets. 2012).

You can learn more about citrulline at the SuppVersity

Citrulline prevents muscle catablism more than leucine

Arginine & citrulline for blood lipid control

EAA, BCAA, or citrulline for anti-catabolism?

Glutamine not citrulline to heal the gut?

Citrulline to ignite fatty acid oxidataion?

High & low dose arginine ineffec- tive NO boosters

On a molecular level fructose has been shown to trigger the production of fat from glucose in the liver (de novo lipogenesis | DNL). It does so by activating certain enzymes via the sterol regulatory element binding protein-1c (SREBP1c) and/or the carbohydrate-responsive element-binding protein (ChREBP). In conjunction with the corollary hepatic oxidative stress and the subsequent increase in insulin resistance, the onslaught of readily absorbed fructose from processed foods and HFCS-sweetened beverages is thus  like gasoline on the fire of the obesogenic baseline diet some people refer to as the "standard american diet" (learn why the "SAD-diet" is so good at making you fat). On the whole, however, the accumulation of fatty streaks in the liver that's so characteristic of NAFLD is yet only the point of departure of the journey to the land of the super-obese type II diabetics.


Now this journey from slightly overweight to super-obese is a journey of which many previous studies studies already suggested that it could take a very different route if people consumed higher amounts of protein and/or certain amino acids (AAs):

• Theytaz et al. (2012), for example, found a "liver cleansing" increase in VLDL-TG release by the liver with an essential AA-enriched diet, and
• Bortolotti et al. (2012) showed that a protein-enriched diet can effectively reduce the fructose induced lipid accumulation in the liver through increased energy expenditure. 

As Prasanthi Jegatheesan et al. point out, "[t]hese beneficial effects of AAs or proteins may arise through lipid oxidation, decreased DNL, and modulation of genes involved in lipid metabolism" (Jegatheesan. 2015). Since citrulline is the precursor for the renal synthesis of Arg, which is known to improve insulin sensitivity and lipid metabolism, and has been shown to have beneficial effects on the level of plasma triglycerides and fat deposition in the liver, the authors of a recent study speculated that "Cit supplementations might [...] able to limit the development of fructose-induced NAFLD" (Jegatheesan. 2015). Morever, Jegatheesan et al. expected to see similar effects with other nonessential amino acids (NEAA), of which their own previous research had shown that they may offer similar anti-NAFLD effects.

Where's the control group? Previous studies show that diets which are supplemented with NEAAs (alanine, glycine, proline, aspartate, histidine, and serine) or citrulline have metabolic and nutritional effects similar to a regular control diet, alone (Osowska. 2006; Jegatheesan. 2015). The CNEAA group is thus the "control" group in the study at hand. That's "ok" and doesn't make the study results useless, but in view of the fact that the data in Figure 2 shows more than just an ameliorative effect of citrulline on NAFLD, I would have preferred a regular control group in which the rodents had been fed standard chow without added non-essential amino acids.

To confirm or falsify their hypotheses, the researchers randomized twenty-two rats into four groups on different diets:

• CNEAA as in control - control diet without added fructose + 1g/kg non-essential amino acids (for humans that's roughly 11g per day | this was the control diet in the study at hand)
• F as in fructose- control diet enriched with 60% fructose without supplements
• FNEAA as in control + fructose - fructose enriched diet (F) + 1g/kg non-essential amino acids (which happens to be the control diet in the study at hand)
• FCIT as in fructose + citrulline - fructose enriched diet (F) + 1g/kg citrulline

In that, it's important to note that the NEAA supplement contained isomolar amounts of the 6 AAs and was isonitrogenous to the Cit diet. So, a mere difference in the nitrogen content of the chow cannot explain the obvious differences that occurred over the course of the 8-week study period.

Figure 1: Relative changes in liver weight, hepatic triglyceride content as well as the liver markers AST, ALT and ALP a marker of kidney health  compared the "control" group (CNEAA | Jegatheesan. 2015)

A period, in which the rodents in the fructose enriched diet group (F) developed NAFLD. A fate the rats in the FCit and the FNEFA group did not share - even though the amount of fructose in their diets was exactly as high as it was in the F group.

Figure 2: Both FNEAA and FCit rodents had a better body composition than the rodents on the NEAA supplemented control diet, but the differences reached statistical sign. only compared to the fructose (F) group (Jegatheesan. 2015)

In that, it is unquestionably worth noting that we are not talking about a mere amelioration of the fructose induced damage. If you look at the data you will notice that the rodents with the alanine, glycine, proline, aspartate, histidine, and serine enhanced fructose enriched diets actually ended up having healthier livers than those on the non-fructose diet... if that's not convincing evidence that the commonly heard, and painfully overgeneralized claim that "fructose is the root cause of all metabolic diseases" is bogus, I don't know.

So, why would you even consider citrulline, if the NEAA combo is better for your liver? 

Well, the reason that the average physique enthusiast, may still choose citrulline as his "fructose buffer" of choice is easy: Firstly, the differences in terms of liver health are not really statistically significant. Secondly and more importantly, though, citrulline triggered a reduction in visceral and total fat mass and a relative increase lean mass that was not observed in the NEAA group. And let's be honest: Isn't this type of body recompositioning effect what many of you are striving for?

What is most astonishing though, is that you could have these fat loss and muscle gain effects not just despite, but maybe even because you're guzzling HFCS drinks all day (obviously we'd have to have a citrulline + baseline diet group to confirm that). If we assume that the results translate 1:1 to human beings, the one thing you had just ~10g of citrulline per day. Is this possible? Well, it is, but let's be honest with ourselves: The inter-group differences between the control and the citrulline + fructose were not statistically significant. So while there were improvements those were not pronounced enough to be of statistical significance even in rodents. It is thus not really surprising that you haven't heard of citrulline as the "get jacked" amino acid very often... even though, evidence that it can help you to get jacked does exist (more).

Bottom line: It is quite astonishing how commonly ignored correlates of high fructose intakes can turn an obesogenic liver killer into a regular energy supplier. I mean, look at the data in the study at hand: Where's the evidence that fructose is worse than any other energy source, when a simple increase in NEAA or citrulline intake does not just nullify its effects but has the rodents on the 60% fructose diet end up leaner and with lower liver fat and better AST and ALT levels than their peers on the control diet (these differences are only partly statistically sign., though).

Citrulline & Glutathione - GSH Amplifies & Prolongs CIT's NO Boosting Effects During + After Biceps Workout | learn more.

So, just as Jegatheesan et al. say: When combined with NEAAs or citrulline, fructose is not just harmless, but can even "produced an overall change in nutritional and metabolic status, with lower body weight and altered body composition, [in spite of identical" food/energy [...] among groups" (Jegatheesan. 2015). Unfortunately, the precise mechanisms involved still need to be investigated. Jegetheesan et al. are yet relatively convinced that NEAAs and citrulline act via different pathways: "NEAAs may act through GCN2, citrulline could act on the liver via PPARa and the down-regulation of SREBP1c, for example, via protein kinase B and mTOR pathway, but also via the improved insulin sensitivity enabled by peripheral Arg bioavailability" (ibid). Just as it is the case for the applicability in humans, though, these hypotheses require future experimental verification | Comment!

References:

• Bortolotti, Murielle, et al. "Effects of dietary protein on lipid metabolism in high fructose fed humans." Clinical Nutrition 31.2 (2012): 238-245.
• Jegatheesan, Prasanthi, et al. "Effect of specific amino acids on hepatic lipid metabolism in fructose-induced non-alcoholic fatty liver disease." Clinical Nutrition (2015).
• Jegatheesan, Prasanthi, et al. "Citrulline and Nonessential Amino Acids Prevent Fructose-Induced Nonalcoholic Fatty Liver Disease in Rats." The Journal of Nutrition (2015): jn218982.
• Marchesini, Giulio, et al. "Nonalcoholic fatty liver disease a feature of the metabolic syndrome." Diabetes 50.8 (2001): 1844-1850.
• Osowska, Sylwia, et al. "Citrulline modulates muscle protein metabolism in old malnourished rats." American Journal of Physiology-Endocrinology and Metabolism 291.3 (2006): E582-E586.
• Theytaz, Fanny, et al. "Effects of supplementation with essential amino acids on intrahepatic lipid concentrations during fructose overfeeding in humans." The American journal of clinical nutrition 96.5 (2012): 1008-1016.
• Volynets, Valentina, et al. "Nutrition, intestinal permeability, and blood ethanol levels are altered in patients with nonalcoholic fatty liver disease (NAFLD)." Digestive diseases and sciences 57.7 (2012): 1932-1941.

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.

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 ;-)

L-Citrulline Improves Sexual Performance: 50% Chance of Improvement with just 1.5g/day

You probably receive the same amount of daily spam mails trying to refer you to dubious viagra retailers somewhere on the Cayman Islands, or wherever else in the world. In case, you ever even thought about ordering stuff like that, you may as well consider getting some l-citrulline from your local supplement store first.

Scientistsfrom the University of Foggia in Italy (Cormio. 2011) have found that supplementation with as little as 1.5g/day l-citrulline improved erection hardness in men with mild erectile dysfunction (and I always thought Italian men would not have any erection problems ;-):

Significant improvement in the EHS [erection hardness score] from 3 to 4 was reported by 2 (8.3%) of the 24 men when taking the placebo and 12 (50%) of the 24 men when taking L-citrulline (P < .01). The mean number of intercourses per month, a nonvalidated patient-reported outcome we have commonly used in patients treated for ED, was 1.37 ± 0.93 at baseline, 1.53 ± 1.00 at the end of the placebo phase (P = .57), and 2.3 ± 1.37 at the end of the treatment phase (P < .01). For treatment satisfaction, all 12 patients reporting an EHS improvement from 3 to 4 scored very satisfied, and the remaining 12 scored not satisfied and all asked for a PDE-5 inhibitor prescription at the end of the study phase. Finally, no AEs [adverse events] occurred.

So, this is a 50% chance of improvement. In view of the price of l-citrulline (as bulk-powder) it would be worth a try. I promise, I won't tell anyone ;-)

L-Citrulline Against Arterial Stiffness

You probably know it from the ingredient list of your pre-workout or amino acid product, l-citrulline. Being a possible precursor of l-arginine it is included in those formulas to increase nitric oxide levels and deliver those "skin bursting pumps" the advertisements brag about.

In a recent study, scientists from Japan (Ochiai. 2010) found that 1 week of l-citrulline supplementation at 5.6g/day effectively reduced arterial stiffness in 15 healthy males:

Compared with the placebo group, baPWV [index of arterial stiffness] was significantly reduced in the l-citrulline group (p<0.01). No significant differences in blood pressure (BP) were found between the two groups, and no correlation was observed between BP and baPWV. The serum nitrogen oxide (NOx, the sum of nitrite plus nitrate) and NO metabolic products were significantly increased only in the l-citrulline group (p<0.05). Plasma citrulline, arginine and the ratio of arginine/asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase (arginine/ADMA ratio) were significantly increased in the l-citrulline group compared with the placebo group (p<0.05, p<0.01, p<0.05, respectively). Moreover, there was a correlation between the increase of plasma arginine and the reduction of baPWV (r=-0.553, p<0.05).

Beyond the reduction in arterial stiffness, these results confirm the hypothesis that l-citrulline effectively increases NO metabolism (probably even more than l-arginine) AND, what's even more important, reduces an endogenous inhibitor of NO synthase, so that more nitric oxide may be produced.