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 ;-)

Caffeine Protects Brain Function Against Stress & SAD Diet; Coffee Withdrawal, Anxiety & More; Giardia, Messy Subtenant W/ Gusto For Arginine; Vit B6 & n6:n3 PUFA Ratio

19 Billion Euro that's the estimated 2011 financial burden due to lung cancer, alone, here in Europe and the On Short Notice figure of the week (information based on ESMO2012 press release)

Those of you who are also following the SuppVersity facebook news, will probably recognize the figure on the right: 16,000,000,000€ or $24,419,000,000, that's the estimated economical burden due to lung cancer, alone, here in Europe (cf. "Who cares if people are dying as long as the economy is thriving?"). An enormous financial loss, and still not the reason that this is my figure of the week. Rather than the financial damage, itself, it is the tragic fact that only the latter, yet not the fate of the patients and their families, would make a valid argument, when policy makers were debating a long overdue, total and all-encompassing public smoking ban... but now for a couple of more sciency, yet not less intriguing news from the past week.

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Problems thinking straight? Guess what: 3-4 cups of coffee could help :-) According to a soon-to-be-published paper by scientists from the Jordan University of Science and Technology in Irbid, Jordan, the ingestion of the human equivalent of approximately 3.8mg caffeine per kg body weight or 3-4 cups of coffee per day, can inhibit both, the stress, related as well as diet induced (we are talking of the "typical" Western diet (WD), that's both high in carbohydrates and fat) cognitive impairments (Alzoubi. 2012)... well, at least in the researchers 3-months rodent study it worked like a charm

• learning trial: animals in the caffeine/stress, caffeine/WD, and caffeine/stress/WD groups made fewer errors, than non-supplemented stressed or WD animals; overall their performance was comparable to those of the control
• memory tests: treatment reduced the number of error and restored short-term memory and long-term memory during chronic stress and/or WD (P < 0.05) to normal levels

With respect to the underlying mechanisms the scientists speculate that caffeine may "act mainly by inhibiting adenosine receptors" (Alroubi. 2012), which has in turn been shown to to inhibit long term potentiation (LTP) in rat hippocampal slices and disrupt the process of learning and memory at the synaptic level by blocking release of glutamate (de Mendonca. 1994).

Additionally, caffeine has also been shown to increases the expression of hippocampal brain-derived neurotrophic factor (BDNF) and its receptor, which is impaired in response to chronic stress and a hypercaloric Western diet (Aleisa. 2006; Molteni. 2004) and leads to deteriorations in cognitive performance. In the long run those effects could also contribute to the anti-dementia and anti-Parkinson's effects, I mentioned in the recent SuppVersity post on the insulin sensitizing effects of coffee.

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Figure 1: While the Hedonic tone and alertness reduced to baseline on day 5 of caffeine withdrawal, the habitual caffeine consumers had >15% higher anxiety scores on day 7 after giving up on their daily dose of methylxanthine (data calculated based on Smith. 2012).

Don't worry, caffeine will also work for humans. And what's best, upon short-term withdrawl (8 days) your cognitive performance is not going to suck - at least not as much as when you are stressed or living on pizza and French fries, only. All that and a couple of interesting other results have been published ahead of print in the online version of the Journal of Pharmacology (Smith. 2012).

To probe the effects of acute caffeine ingestion on cognitive performance and the influence of previous caffeine consumption and withdrawal, Andrew P Smith, Gary Christopher and David Sutherland recruited 70 volunteers (25 male, 45 female; mean age 22.8 years). The 35 consumers (>100mg caffeine /day, mean 300mg; range 110–600 mg) were put on withdrawal and tested on day 2, alone and without caffeine, and day 8 together with the non-consumers in a double-blind placebo-controlled fashion. During the caffeine challenge, the cognitive performance was tested twice, once before and once 30min after the provision of the caffeinated beverages.

Anxious, but smart: Caffeine gives you the edge

The results of the trial clearly indicate that the ingestion of 2 mg/kg of caffeine, which were served in decaffeinated coffee or tea 30min before the testing procedures, were associated with faster simple reaction times, fewer long responses, greater detection of targets in the cognitive vigilance task, and faster encoding of new information.

"The results confirmed previous findings, with ingestion of caffeine being associated with a faster simple reaction time, fewer long responses, more targets detected and faster encoding of new information. There were no main effects of consumer status, nor were there any significant interactions between caffeine and consumer status." (Smith. 2012)

Notwithstanding, I believe that many of you will probably be more interested in the effects of caffeine withdrawal on overall withdrawal symptoms (figure 1, top), as well as the alertness, hedonic tone and anxiety (figure 1, bottom) and the cognitive performance on day 2 of the withdrawal period (figure 2, left), than in any of the well-established performance cognitive performance boost, right?

Figure 2: Performance on day 2 of withdrawal phase (w/out caffeine) and on day 8 before (w/out caffeine) and after (w/ caffeine)the ingestion of decaffeinated tea or coffee with 2mg/kg caffeine in it (data based on Smith. 2012)

As you can see on the left-hand side of figure 2 there was a minimal performance decline on day 2 of the withdrawal phase, but the latter was statistically not significant and all measured markers of cognitive function had returned to normal on day 8 (remember longer response times = worse performance!), when the resumption or first time provision of caffeine spiked the reaction times and lowered the mistakes in all tests, irrespective of whether the subjects were former habitual consumers on withdrawal, or not.

Outside of controlled experiments "real" coffee and tea do at least as well

Since a large cup of coffee contains about the same amount of caffeine the scientists simply added to decaffeinated beverages, to ensure that the drinks could not be distinguished (by their smell for example), you can simply stick to your regular coffee and if you want to enjoy similar benefits. And to be honest, in view of the plethora of benefits of chronic low dose coffee consumption, I would not even think for a second about whether or not you may be missing out on the occasional boost, when you are not "going on withdrawal" from time to time...

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Figure 3: W/out arginine (Arg-) intestinal epithelial cells can't proliferate (graph based on Stadelmann. 2012)

Giardia eats away your guts arginine supply and makes itself at home within an increasingly morbid digestive tract! As a group of scientists from Sweden and Argentina reports in their latest paper, the protozoan parasite, Giardia intestinalis, feasts on the arginine your gut cells need to proliferate (Stadelmann. 2012). This will lead to reduced polyamine levels and upregulated cell cycle inhibitory genes, which will eventually disrupt the the cell cycle of the intestinal epithelial cells. The reduced intestinal epithelial cell proliferation, on the other hand, allows the gut pathogen to thrive and will, in the long run, disrupt the intestinal tissue homeostasis and thus initiate the decay of the intestinal epithelium  - a central feature of so many of the wide-spread gut pathologies.

Provision of additional arginine + citrulline can help ... in the short run

Now, the good news about all that is that the in-vitro data in figure 3 clearly suggests and anecdotal, as well as the effective therapy of diarrhea patients with arginine/citrulline actually confirm that the provision of supplemental arginine (or citrulline) constitutes a cheap and readily available way to ameliorate the decay, until the bugs have been eradicated by antimicrobial drugs.

A pros pos, antimocrobial drugs, with regard to latter, Noa Tejman-Yarden and Lars Eckmann write in a recent review of the latest drug innovations, that despite the fact that metronidazole and other antimicrobials are usually effective, "treatment failures are common and antimicrobia resistance occurs" (Tejman-Yarden. 2011), so that it would appear as if complex derivatives of 5-nitroimidazole and benzimidazole, which form the core structure of the most widely used antigiardial drugs, will replace them in the short-run. At least for so long, until several new classes of antigiardial drug candidates that have already been identity by high-throughput screening of large compound libraries, will eventually hit the market (Tejman.Yarden. 2011)

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More about vitamin B6: Helps with neurotransmitters synthesis; is involved in nerve function and necessary for normal brain development & function; influences mood, and melatonin production; effects circadian clock; is needed for B12 absorption and thus red blood cell production

When low: "Pins and needles" in extremities, mental disorders, seborrheic dermatitis, estrogenic PMS, dizziness, irritability, kidney stones, abnormal EEG, anemia, convulsions, edema (water retention), hypothyroidism, migraine-headaches, glossitis, lymphopenia

When high: Depression, suicidal tendencies, severe fatigue, mood swings, low blood sugar, migraine-headaches, heart palpitations, thyroid abnormalities (hyper- in the short, hypo in the long term), numbness in hands and/or feet, spinal / nerve degeneration, muscle spasms / cramps, osteoporosis, arthritis, higher blood pressure (short-term suppl.), lower blood pressure (long-term suppl.), mineral imbalances (high phosphor & magnesium vs. low sodium & calcium), restlessness, insomnia, vivid dreams, decreased estrogen & prolactin, depressive PMS.

RDA (adults): 1.3 mg*
*higher for pregnant women & >50y

Upper tolerable limit: 30-100mg*
*depending on the source of information

Food sources: chicken, turkey, tuna, salmon, shrimp, beef liver, milk, cheese, lentils, beans, spinach, carrots, brown rice, bran, sunflower seeds, wheat germ, and whole-grain flour

n6:n3 ratio does not depend on dietary intake alone: A marginal deficiency in vitamin B6 will skew your serum PUFA levels towards the N6-side That's the long and short of the results of a study that's going to be published in the October issue of the Journal of Nutrition.

Mei Zhao and her colleagues analyzed the fatty acid profiles in plasma, erythrocytes, and peripheral blood mononuclear cells (PBMC) of healthy men and women who had been fed a low-vitamin B-6 (pyridoxine) diet for 28 days and observed that contrary to the plasma HDL and LDL cholesterol concentrations, the amount of free fatty acids (FFA) in the blood and the erythrocyte and PBMC membrane fatty acid compositions, neither of which showed any statistically significant changes, the amount of all long-chain polyunsaturated fatty acids, i.e. arachidonic acid (n6) and EPA and DHA (n3) decreased from 548 ± 96 to 490 ± 94 μmol/L, 37 ± 13 to 32 ± 13 μmol/L, and 121 ± 28 to 109 ± 28 μmol/L, respectively.

The subsequent 8% increase in the total n6:n3 PUFA ratio from 15.4 to 16.6 is not alarming, but if this trend would continue linearly, it would certainly become problematic, in the long run. Moreover, the decrease in both n6 and n3 long-chain PUFAs (of which people tend to forget that the "inflammatory" arachidonic acid is as vitally important as its "anti-inflammatory" omega-3 counterparts) could provide an alternative / complementary mechanistic explanation for the increased cardiovascular disease risk that has been associated with vitamin B-6 deficiency.

In view of the fact that the RDA is not exactly high and can easily be achieved from dietary sources, along (as long as you follow a diversified whole foods diet), and considering the fact that high levels of B6 have been associated with more negative side-effects than B6 deficiency (see infobox on the right; please note that I collected the information on a couple of trustworthy websites on RDAs & co and did not verify the research on each of them!), I would however caution against the typical Western "more helps more" supplementation mentality.

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Figure 4: Easy come, easy go - the mass you gain and the fat you lose by doing nothing than simply injecting testosterone is lost / regained within 6 months after discontinuation of the "testosterone therapy" (Forbes. 1992); read more about the role of testosterone in skeletal muscle hypertrophy in the Intermittent Thoughts on Building Muscle

In view of the fact that (a) today's short news items are pretty long(ish) and you still got a couple of interesting facebook news to check out, such as...

• health related: "Vitamin D: We probably won't know before 2017, whether supplementation is advisable", "Aromatase inhibitors to treat endometriosis", or 
• muscle related: "The muscle sparing effects of CLA + Omega-3" and a true classic "Muscle mass gained on injectable testosterone is lost, when injections are seized", 

... and a plethora of additional gems from the realms of health, exercise, nutrition & supplementation, I will call it a day for today and save the exercise and a couple of other exciting On Short Notice items for later next week.


References:

• Aleisa AM, Alzoubi KH, Gerges NZ, Alkadhi KA. Chronic psychosocial stress-induced impairment of hippocampal LTP: possible role of BDNF. Neurobiology of Disease 2006;22:453–62. 
• Alzoubi KH, Abdul-Razzak KK, Khabour OF, Al-Tuweiq GM, Alzubi MA, Alkadhi KA. Caffeine prevents cognitive impairment induced by chronic psychosocial stress and/or high fat-high carbohydrate diet. Behav Brain Res. 2012 Sep 20.
• ESMO. Press releases related to the ESMO 2012 Congress of the European Society for Medical Oncology in Vienna.
• Forbes GB, Porta CR, Herr BE, Griggs RC. Sequence of changes in body composition induced by testosterone and reversal of changes after drug is stopped. JAMA. 1992 Jan 15;267(3):397-9.
• de Mendonca A, Ribeiro JA. Endogenous adenosine modulates long-term potentiation in the hippocampus. Neuroscience 1994;62:385–90.
• Molteni R, Wu A, Vaynman S, Ying Z, Barnard RJ, Gomez-Pinilla F. Exercise reverses the harmful effects of consumption of a high-fat diet on synaptic and behavioral plasticity associated to the action of brain-derived neurotrophic factor. Neuroscience 2004;123:429–40.
• Smith AP, Christopher G, Sutherland D. Acute effects of caffeine on attention: a comparison of non-consumers and withdrawn consumers. J Psychopharmacol. 2012 Sep 19.
• Stadelmann B, Merino MC, Persson L, Svaerd SG. Arginine Consumption by the Intestinal Parasite Giardia intestinalis Reduces Proliferation of Intestinal Epithelial Cells. PLoS ONE. 2012; 7(9): e45325. 
• Tejman-Yarden N, Eckmann L. New approaches to the treatment of giardiasis. Curr Opin Infect Dis. 2011 Oct;24(5):451-6.

8g/day Citrulline Increase Leg Workout Performance - More Reps on Leg Press, Hack Squat & Leg Ext. in Exp. Gymrats

The study tested only leg exercises, but you can safely expect increased reps on other exercises, as well.

Citrulline? Yes that's the stuff the supplement industry claims to be a better version of arginine. A "super pump supplement", but not necessarily an ergogenic - in spite of the fact that corresponding evidence from rodent and human studies (Briand. 1992; Pérez-Guisado. 2010; Giannesini. 2011) existed years before the study at hand was published (Wax. 2014).

Benjamin Wax and his colleagues from the Mississippi State University and the Auburn University  investigated the effects of citrulline malate supplementation on lower-body resistance exercise performance, blood lactate, heart rate, and blood pressure.

Based on citrulline malate’s chemical composition and a review of the current literature Wax et al. hypothesized that citrulline malate supplementation would mitigate fatigue occurring to the working muscle; therefore, augmenting resistance training performance.

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

To test their hypothesis the researchers recruited 12 advanced resistance trained male subjects (85kg body weight; <12% body fat; 22.1 ± 1.4 years) and conducted a randomized, counterbalanced, double blind study.

The subjects were randomly assigned to placebo or citrulline malate (8 g; 60 minutes before the workout) groups and then performed repeated bouts of multiple lower body resistance exercise:

"Subjects warmed up on an upright stationary bike (Life Fitness, Brunswick Corporation, Lake Fores, IL) for five minutes, at 60 – 70 revolution/minute with a mass of 3 – 5 kg. Following this warm up, subjects performed two warm up sets (10 repetitions at 90.9 kg and 8 repetitions at 136.4 kg) on the leg press machine. Subjects rested three minutes between sets during the warm up and trial sets.

Next, 60% of each subject’s predetermined 1RM was loaded on the leg press machine and the subject completed as many repetitions as possible until failure occurred. This process was completed for 4 additional sets for a total of 5 sets on the leg press. Next, the subjects performed one warm upset (10 repetitions) on the hack squat machine at a mass of 40.9 kg. This warm up set was followed by 5 sets of 60% of their predetermined 1RM to failure. Finally, following one warm up set (10 repetitions at 36.4 kg) on the leg extension, subjects completed 5 sets of 60% of their 1RM to failure." (Wax. 2014)

The rest periods (recovery periods between sets of exercise), exercise order, and number of sets performed were the same for all subjects in this investigation, for sessions 2 and 3. Blood lactate, heart rate, systolic blood pressure, and diastolic blood pressure were determined pre and post exercise.

Practical applications - What the scientists say: "Although citrulline malate supplements are marketed to improve muscle performance via a reduction in lactic acid and ammonia production, the current study does not fully support this assertion. While our investigation did note improved muscle performance occurring during the strength protocol,blood lactate remained indifferent comparing the citrulline malate treatment to the placebo treatment. The known capacity of citrulline malate to increase plasma L-arginine (Hickner. 2006), act as a buffer to lactate and hyperammonemia (Briand. 1992; Giannesini. 2011; Verleye. 1995) remain valid; however, further research is necessary to determine which mechanism may be directly attributed ergogenic effects occurring during resistance training protocols. Finally, specific investigations utilizing training protocols designed to test muscular strength and power are warranted." (Wax. 2014)

The exercise protocol resulted in sequential significant (p < 0.05) decrease in the number of repetitions in all three exercises. However, subjects in the citrulline malate group performed significantly (p < 0.05) higher number of repetitions during all three exercises compared to placebo group.

Figure 1: Wax et al. observed significant increases in maximal leg press, hack squat, and leg extension (not shown) repetitions in response to the ingestion of 8g of citrulline malate 60min before exercise (Wax. 2014)

Blood lactate and heart rate, on the other hand, were significantly increased (p < 0.05) post-exercise in both groups with no significant inter-group differences between citrulline malate and placebo (p > 0.05).

Bottom line: I am not sure, if the study at hand is going to change anyone's perspective on citrulline. The "pump" is after all (for whatever reason) still what appears to be most attractive to trainees. The fact that the increased number of reps could translate into increased strength and size gains over time, on the other hand, doesn't appear sexy enough to be marketed as the main benefit of citrulline.

Can citrulline prevent muscle loss, when you're dieting | learn more

Apropos "main effect", there I guess you will remember that citrulline will also affect protein synthesis, right? I've written about these effects in August last year in my article "Citrulline = The Dieter's Amino Acid? Citrulline Maintains Muscle Protein Synthesis & Strength Endurance During Caloric Deficits Better Than Leucine!?" (read more).

So, if you don't consider increased rep numbers sexy enough, you may feel that a reduced muscle breakdown on your next diet may be worth heading over to the bulk supplier of your choice and order a 1kg bag of citrulline malate for 100$ (will last 125 days) - no? Well, honestly, I am not sure if it's worth that, either | What do you think?

References:

• Briand, Joël, et al. "Use of a microbial model for the determination of drug effects on cell metabolism and energetics: Study of citrulline‐malate." Biopharmaceutics & drug disposition 13.1 (1992): 1-22.
• Hickner, Robert C., et al. "L-citrulline reduces time to exhaustion and insulin response to a graded exercise test." Medicine and science in sports and exercise 38.4 (2006): 660-666.
• Giannesini, Benoît, et al. "Citrulline malate supplementation increases muscle efficiency in rat skeletal muscle." European journal of pharmacology 667.1 (2011): 100-104.
• Pérez-Guisado, Joaquín, and Philip M. Jakeman. "Citrulline malate enhances athletic anaerobic performance and relieves muscle soreness." The Journal of Strength & Conditioning Research 24.5 (2010): 1215-1222.
• Verleye, M., et al. "Effects of citrulline malate on bacterial lipopolysaccharide induced endotoxemia in rats." Arzneimittelforschung 45.6 (1995): E712.
• Wax, Benjamin, et al. "Effects of Supplemental Citrulline Malate Ingestion During Repeated Bouts of Lower-body Exercise in Advanced Weight Lifters." The Journal of Strength & Conditioning Research (2014).

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.

---

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.

Beyond Nitric Oxide II: Arginine & Citrulline Modulate Blood Lipids & Liver Fatty Acid Composition And Ameliorate Aortic Lipid Deposition in High Fat Fed Rats

Image 1: The mouse on the left probably
had too little arginine in its high fat diet
(just kiddin' ;-)

This is a follow up not on yesterday's third, but on the second installment of the Amino Acids for Super Humans series on Carl Lenore's Super Human Radio. In that episode I had already alluded to the metabolic benefits of arginine and citrulline supplementation, which reach far beyond their purported use as ergogenic aids and nitric oxide booster. A very recent study (El Kirsh. 2011) published a week ago ahead of print in the online issue of Cell Biochemistry & Function confirmed and expanded on the results of previous studies which showed beneficial effects on parameters of organ (specifically heart and liver) in rodent models.

6 groups of rats fed either a normal diet or the dubious "high fat" diet, scientists use to induce obesity along with the characteristic symptoms of the metabolic syndrome (insulin resistance, arteriosclerosis, etc.) were fed one of the following diets:

• group 1: normal food, no supplement
• group 2: normal food, arginine (100mg/kg; human equivalent 16mg/kg)
• group 3: normal food, citrulline (100mg/kg)
• group 4: high fat diet (HFD), no supplement
• group 5: high fat diet (HFD), arginine (100mg/kg)
• group 6: high fat diet (HFD), citrulline (100mg/kg)

Both the negative effects of the high fat diet, as well as the ameliorative effects of arginine and citrulline on serum transaminase levels (indicators of liver "activity" /high levels are mostly interpreted as signs of liver damage), triglycerides, cholesterol levels and indices of atherosclerosis were profound:

HFD feeding increased significantly serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, urea and all lipid profiles and decreased significantly serum high-density lipoprotein-cholesterol (HDL-c) and non significantly serum nitric oxide levels. L-arginine or L-citrulline administration reversed the increase in serum AST and ALT activities, urea and all lipid profiles.

As it can be seen in figure 1, the positive effects of arginine and citrulline even went beyond the mere amelioration of the detrimental effects of the high fat diet by increasing HDL-c and nitric oxide levels, decreasing the count of dangerous V-LDL particles even compared to normal diet and modulating the relative percentages of 18 : 0, 20 : 0 and 22 : 6 to 16 : 0 fatty acids in the livers of the rats on the high fat diet.

Figure 1: Triglyceride and cholesterol levels of rats fed normal chow (control) and rats on a high fat diet (HFD) supplemented with either arginine (HFD+Arg) or citrulline (HFD+Cit) (data adapted from El Kirsh. 2011)

Moreover, the beneficial effects on heart health that are generally ascribed to such improvement in blood and tissue markers could actually be confirmed by light and transmission electron microscopic images of the thoracic aorta. While the HFD group that did not receive supplemental arginine or citrulline "demonstrated structural changes in the endothelial cells of the intimal layer, medial smooth muscle cells as well as in the adventitial layer", there was only "little structural alterations in animals supplemented with L-arginine or L-citrulline along with HFC feeding".

Taken together, these and the results I reported in the aforementioned episode of Amino Acids for Super Humans should well be able to change your perspective on your favorite pre-workout product forever - even if it does not give you the pump the fancy ad you saw when you bought it promised, it may well save your organs from the consequences of one or another dietary faux pas. I hope, I do not have to tell you, though, that it would border on or rather be plain nutritional idiocy, if you used arginine and citrulline to make up for the consumption of the famous westernized high fat, high carb, low protein fast food diet (which would be similar to the HFD the rats consumes)!

Amino Acids for Super Humans. Part II - Arginine, Citrulline and Ornithine: Nitric Oxide and Beyond

This relevant for all of you - regardless of whether you can or cannot spare the time: Tune in live and listen to me @ Carl Lenore's Super Human Radio to learn that amino acids are far more than just the building blocks of the proteins of your body...

Listen live to SHR @ 12:00PM ET

Amino Acids for Super Humans. Part II
(update: click here to download podcast)
Arginine, Citrulline and Ornithine: Nitric Oxide and Beyond

In the second installment of the show we are going to look at the purported and busted NO Booster l-Arginine, its metabolites citrulline and ornithine, malic acid or alpha ketoglutarate binding of this amino acids and their largely overlooked effects on glucose homeostasis, protein synthesis and detoxification.

As usual, I will do my best to provide relevant examples and relate the theory to practical advice. The magic of individual amino acids will be tackled in the shows to come.

Update: The show is now available for download.

High and Low Dose of L-Arginine Equally Ineffective in Promoting Nitric Oxide Synthesis in Young Active Men at Rest - GH and IGF-1 Response Negligible.

Image 1: Arginine based first generation
"nitric oxide booster", such as BSN's
NO-Xplode still have a huge fanbase

"No NO from l-arginine", you probably remember this or similar headlines from a few years ago, when the first studies seemed to indicate that l-arginine, the main "working" ingredient in all the (over-)hyped nitric oxide (NO) boosters that had become an integral part of the line-up of each and every major supplement producer was found not to increase the amount of nitric oxide in their consumers' bloodstream. Although these results stand in stark contrast to the early studies using intravenous infusion of l-arginine, which established the myth of the NO and growth hormone boosting effects of 2-amino-5-guanidinovaleric acid (l-arginine),  a 2007 study by Malinauskas et al. (Malinauskas. 2007) found that 8% of the male student athletes who participated in their survey were still interested in the ingestion of l-arginine containing supplements. Thus, it does not come as a surprise that - even with the rise of the so-called 2nd generation pre-workout products - the classic, l-arginine based products such as NO XPlode, SuperPump and Co. still have a huge fanbase, the majority of whose members, asked about why they are taking the respective product, will certainly answer "for the pump".

A very recent study by Scott C. Forbes and Gordon J. Bell , published in the Journal of Appl. Physiol. Nutr. Metab. (Forbes. 2011), is yet only the latest in series of investigations, which puts into question, how much of a placebo effect may be involved in the real-world observations of "enormous pumps and vasolidation" many consumers of l-arginine based preworkout products report. 14 weight training (4 sessions per week) male participants, who had been free from other nutritional supplements for at least 12 weeks participated in the study. In a double-blind repeated measure design with 3 testing conditions (baseline, placebo/l-arginine, l-arginine/placebo) that were separated by 7 days, the subjects received either high or low dose (0.075 (low) or 0.150g/kg (high)) l-arginine or placebo (flour) solved in 500ml of water after a 10 hour fast. Interestingly,

plasma concentrations significantly increased (p < 0.05) to a similar level [+178% and +204%] above resting values, respectively)at all time points in both the low- and high-dose conditions, while there was no change in the placebo condition over time.

Or, in other words, there was a tight regulation of maximal l-arginine levels in the plasma of the subjects, either via decreased absorption or increased metabolism of the orally ingested l-arginine (it is estimated that with increasing doses >40% of the ingested arginine is degraded by the small intestine). 

Apart from the issue of gastro-intestinal distress, simply increasing the dosage will thus not further increase the availability of arginine in the blood stream, which at the concentration that were observed in this study also failed to induce "a significant main effect for insulin (p < 0.05)". Previous studies using intravenously administered arginine had shown increased insulin secretion and, in some cases, onset hypoglycemia. In absence of these insulinogenic effects, however, there were also

no significant difference over time or between conditions for GH (Fig. 3), IGF-1 (Fig. 4), or glucose, (p > 0.05);

all that, despite the fact that the area under the curve (iAUC), a measure for the total amount of l-arginine that was taken up and metabolized by the subjects, did in fact reveal "a significant difference between the L-arginine conditions and placebo and between the high and low relative doses." With regard to the nitric oxide production, this dose dependent increase in plasma arginine levels turned out to be useless, or in fact counterproductive, as contrary to expectation the  area under the curve (iAUC) "for NOx with the high relative dose was significantly lower than that for the low dose of L-arginine" - a clear sign that "overuse" of large amounts l-arginine could diminishes the already negligible on nitric oxide production at rest.

Listen live to SHR tomorrow, Thursday @ 12:00PM ET

Before totally discarding arginine, however, I suggest you tune in to tomorrow's episode of Super Human Radio. In the 2nd installment of the "Amino Acids for Super Humans" series, I will provide further insights into what we really know about arginine and its "metabolic children" ornithine and citrulline and how they may effect how you look, feel and perform in and outside the gym.

Citrulline as Substrate Switch. Galactose as Workout Fuel, Glycogen Repletion Not Urgent, 2x a Day 6x a Week = Too Much For Your Antioxidant System, Astaxanthin For IgA

Actually it's not the burn during the workout that matters, but I don't have to tell you that, do I? (pic i-am-beast.com)

What do you do with a whole host of interesting exercise-related nutrition news that are piling up in your archive, but are too good to be "burned" as short links with one sentence of text on the SuppVersity Facebook Wall?

Right! You compile all those news into a potpourri, attach the label "SuppVersity News Potpourri" to it and blow them out in a blogpost of their own. A post that covers the whole peri-workout window as well as the short-/long-term effects on exercise on your anti-oxidant and immune system.

Sounds good? Well, then go ahead...

• Immediate post-workout glycogen repletion in endurance athletes probably useless (Carlsohn. 2013) While the hormonal response and the long-term effects of running around with depleted glycogen levels are a totally different animal, the latest research from the University Outpatient Clinic Potsdam in Potsdam, Germany, clearly suggests that the immediate post-run glycogen-repletion with 1.5g/kg body weight of fast acting carbs per hour is useless...
  
  

  

  Do you remember my "Glycogen-free muscle growth" post(s) from 2011?

  ...at least with respect to the 5,000m running performance of the twelve recreational runners (4m/8w; 1.73 ± 0.11 m, 69.1 ± 13.4 kg). who were involved in Carlsohn et al.'s study.
  

  "Running time during 5,000-m time trials did not differ between bTT (1,305 ± 140 s), following CARB (1,276 ± 125 s) or PLA (1,285 ± 124 s, p= .85). There were no differences in RPE (bTT 18.3 ± 0.3, CARB 18.7 ± 0.3, PLA 18.8 ± 0.9; p= .48), bLa/min, PLA 187 ± 3 beats/min; p= .96).

  In view of these results it should actually not necessary to formulate a "bottom line", but alas...
  

  ---

  Bottom line: "[T]he rationale of recommending immediate carbohydrate intake following exhausting exercise to 5,000-m runners might be questioned" (Carlsohn. 2013). Please keep in mind though that not repleting your glycogen stores at all is not an option - the myth that's been partially busted by the study at hand is that you must do that as fast as possible to maintain maximal performance - not that you must do it at all. 

• "High" galactose foods ?

  Honey	3.10g
  Fermented yoghurt	1.30g
  Beets, canned, regular pack, solids and liquids	0.80g
  Celery, raw	0.66g
  Cherries, sweet, raw	0.59g
  Bockwurst, pork, veal, raw	0.48g
  Corn, sweet, yellow, canned, whole kernel, drained solids	0.36g
  Beans, navy, mature seeds, raw	0.34g
  Snacks, pretzels, hard, plain, salted	0.22g
  Spices, curry powder	0.21g
  Spices, mustard seed, yellow	0.20g
  Spices, paprika	0.19g
  Babyfood, fruit, plums with tapioca, without ascorbic acid, strained	0.19g
  Spices, ginger, ground	0.19g
  Spices, basil, dried	0.19g
  Kiwi fruit, (chinese gooseberries), fresh, raw	0.17g
  Cereals, oats, instant, fortified, plain, prepared with water (boiling water added or microwaved)	0.16g
  Cheese, mozzarella, whole milk	0.15g
  Spices, cloves, ground	0.15g
  Cheese, parmesan, grated	0.15g
  Spices, oregano, dried	0.15g
  Fast foods, cheeseburger; single, regular patty, with condiments	0.15g
  Plums, raw	0.14g
  Peas, green (includes baby and lesuer types), canned, drained soilds, unprepared	0.14g
  Cereals, oats, instant, fortified, plain, dry	0.13g
  Fish, fish portions and sticks, frozen, preheated	0.13g
  Figs, dried, uncooked	0.13g
  Babyfood, plums, bananas and rice, strained	0.12g
  Egg, whole, raw, fresh	0.11g
  Avocados, raw, all commercial varieties	0.10g
  Crackers, saltines	0.07g
  Snacks, tortilla chips	0.07g
  Egg, white, raw, fresh	0.07g
  Snacks, tortilla chips, nacho cheese	0.07g
  Peaches, raw	0.06g
  Melons, cantaloupe, raw	0.06g

  Galactose as alternative workout fuel (Duckworth. 2013) - A recent study from the Leeds Metropolitan University in the UK demonstrates that
  

  "ingesting a solution containing galactose before and during exercise can positively affect postexercise satiety and energy balance throughout the day, compared to a more readily available and widely consumed form of carbohydrate" (Duckworth. 2013)

  The scientists conclude that based on the observations they made, when they provided nine recreationally active eumenorrheic females (mean age 22y; weight 63.3kg) with either 45g galactose (GI~20) or glucose (GI~89) drinks prior to (300 ml) and at every 15 min during a low intensity steady state jog at 65% of their VO2Peak
  
  Note: I guess, it goes without saying that 45g of galactose this is more galactose than you can stomach from ingesting any "high galactose" foods; see table on the right, data in g/100g).
  The scientists measured the substrate oxidation, postexercise satiety and subsequent energy intake on three occasions (GLU, GAL, placebo) and found that
  • the plasma glucose levels were significantly greater throughout the exercise and in the rest period, when the subjects ingested the glucose drink,
  • there were no differences in carbohydrate oxidation, and
  • perceived hunger was significantly lower throughout the galactose compared to both the glucose and placebo trials
  What may yet be most significant for the average trainee trying to shed some weight is the difference in net energy balance, i.e. the difference between energetic costs of the workout, on the one hand, and the energy intake from the glucose / galactose supplement and the food intake during the post-exercise ad-libitum test lunch and the remainder of the day, which was negative only in the placebo and the galactose trial.

  ---

  Bottom line: If you want to shed some body fat and cannot go without an intra-workout beverage pick galactose over glucose, but do a "test run" before you try that in public - the monosaccharide is notorious for its socially not acceptable effects on the evaporations from your gastrointestinal tract ;-)

"Does the Usefulness of Vitamin E Supplementation Depend on Your Activity Level?" It is possible that only those benefit who are already overtaxing their system and will thus need additional protection (learn more)

• Exercise is stressing, but the long-term results are what's associated with improved antioxidant capacity (Lundström. 2013) The data Lundström et al. have collected in their recent 3-week trial involving fourteen 26-year-old volunteers who performed two "strenuous" (intensity targeted to 75% of VO2max) endurance training sessions per day (6 days a week) does in a way underline the validity of the hormesis hypothesis. Despite the fact that the increase in oxidative stress in response to the the allegedly hefty (for non professional athletes) two-sessions a-day, 6-days a week was not significant, the latter was facilitated / buffered by highly significant declines in the total plasma antioxidant capacity (AO).
  
  However, aside from the fact that the AO levels did not fully return to baseline after the subsequent 4-week recovery period, the most intriguing results of the study at hand is the highly significant negative (meaning "if A is high, B is low") correlation between fat-free mass and oxygen uptake, on the one hand, and oxidation stress, on the other.

  ---

  Bottom line: With both of the former, i.e. fat-free mass and oxygen uptake while you exercise, being hallmark features of physical fitness you cannot increase without working out, the balancing act, every trainee has to master is to find the exact i +1 load of stress that allows for adequate recovery and super-compensation in the time to the next workout / mesocycle.

• Low Immunoglobuli, high cortisol and health While there appears to be a general relation between suppressed sIgA and high cortisol levels, on the one hand, and ill-health effects on the other. The latter is not sports-specific (Volkmann. 2006), and elite athletes are, despite suppressed IgA levels capable of normal responses to novel oral vaccinations, "indicating that mucosal immune mechanisms are intact" (Gleeson. 2000).
  Astaxanthin supplementation can ameliorate minor sIgA dump in athletes (Baralic. 2013) Study shows, supplementation with 4mg/day of astaxanthin can ameliorate the decrease in sIgA (marker of immune health) in young soccer players following 2h of exercise.
  
  There are yet two things you have to consider, when you read studies like these:  (a) Scientific evidence of the significance of immunoglobolin measures is not fully conclusive, and (b) the changes placebo group were not even significant.

  ---

  Note: In view of the fact that "[t]he clinical significance of [immunoglobolin changes] in acquired immunity with acute exercise and training remains unknown" (Walsh. 2011), the scientists' conclusion that "astaxanthin supplementation might serve as a countermeasure to sIgA changes associated with continuous intense training", must be taken with some caution wrt to its real-world benefits. 

• Citrulline shifts substrate utilization towards carbs (Faure. 2013) With this last item in today's Exercise Science Potpourri, we are actually coming back to the an issue that has been in the SuppVersity news pretty regularly as of late: the amount fat / glucose you burn during a workout. I guess, I have made my personal perspective that fatty acid oxidation rates during exercise are hilariously overrated pretty clear. This does yet not stop me from pointing you towards the results of a soon-to-be-published study from the Université Paris Descartes the results of which would suggest that supplemental citrulline could work as a "fuel switch".
  
  

  

  Do you remember the December 2011 SuppVersity news on citrullines anti-catabolic effects (go back!)

   The significant downregulation of oxidative enzymes from the Krebs cycle and mitochondrial respiratory chain, the French scientists observed in a group of male Sprague-Dawley rats, when theyy re-fed them after a 12-week period of dietary restriction with a citrulline supplemented diet (+5g/kg chow and thus equivalent to what human studies have been using) compared to the standard chow with an iso-caloric mix on non-essential amino acids added) would at least suggest that "citrulline supplementatio [...] seems to induce a switch in muscle energy metabolism, from aerobia towards anaerobia" (Faure. 2011).
  
  Now, I did already point out that this is not necessarily a bad thing, but they cannot - as you may speculate now - explain the beneficial effects the original NO-supplement ingredient l-arginine on blood glucose management (learn more), because Faure et al. were able to show that "citrulline action is not direct and is not related to arginine" (Faure. 2013).

  

  Figure 1: Activity of enzymes involved in the oxidation of fatty acids; data expressed relative to baseline levels  on ad-libitum diet (Faure. 2013)

  Against that background another effect that was brought about by the high citrulline diet could yet be even more of a major metabolic disadvantage: The increase in metabolically highly glucoes guzzling unflexible type-IIb fibers (see figure 1; learn more), which has been associated with low / non-existent adiponectin levels by Krause et al. (2008).

  ---

  We have to be careful though, with respect to the interpretation and potential implications of these results. Why? Well, there are actually countless reasons: (a) Human beings are no rodents and normal rodents are no athletes, (b) the potential impact of a higher baseline protein intake or the absence of the calorie restriction before the supplementation period (c) different short (study at hand = 1 week) vs. long-term effects, (d) the possible (beneficial ?) involvement of mTOR, which has been shown to be activated by citrulline in previous trials (cf. SuppVersity Dec 28, 2011 and/or Le Plénier. 2011) (e) the fact that some athletes may benefit from the same shift towards glucose and the relative increase in type IIb fibers (not bodybuilders, though!) (f) ... I could go on with this list, but I guess you will see that there is no reason to panic.
  
  Take the Faure study as further evidence for our lack of understanding of the the complex effects and interplay of nutritional and supplemental amino acids on our health and don't forget to come back to the SuppVersity if you want to keep up with the "state of the art" ;-)

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That's it for today! I hope you enjoyed the "ride" and stay tuned for future exercise, nutrition and health science potpourris - write-ups of which I believe they are a necessary and interesting intermediate between the mini-items on Facebook (don't forget to head over there and check out today's 9+ news items) and the elaborate "regular" SuppVersity articles.

References:

• Baralic I, Đorđević B, Đuričić I, Šobajić S, Stanković I, Dikić N (2013). Salivary IgA response to astaxanthin supplementation in young soccer players. Proceedings of the Nutrition Society, 72, E7.
• Carlsohn A, Heydenreich J, Engel T, Kratzenstein S, Mayer F. Does immediate carbohydrate intake following glycogen-depleting exercise affect next day’s 5000 m time trial performance? International Journal of Sport Nutrition and Exercise Metabolism.  2013; 23(S1 -S15).
• Duckworth LC, Backhouse SH, Stevenson EJ, O’Hara JP. Effect of galactose ingestion before and during exercise on substrate oxidation and subsequent energy intake in females. International Journal of Sport Nutrition and Exercise Metabolism.  2013; 23(S1 -S15).
• Le Plénier, S., Walrand, S., Noirt, R., Cynober, L., Moinard, C., Effects of leucine and  citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway? Amino Acids. 2011.
• Krause MP, Liu Y, Vu V, Chan L, Xu A, Riddell MC, Sweeney G, Hawke TJ.Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function. Am J Physiol Cell Physiol. 2008 Jul;295(1):C203-12. 
• Stuart CA, McCurry MP, Marino A, South MA, Howell ME, Layne AS, Ramsey MW, Stone MH. Slow-Twitch Fiber Proportion in Skeletal Muscle Correlates with Insulin. Responsiveness. J Clin Endocrinol Metab. 2013 Mar 20. 
• Volkmann ER, Weekes NY. Basal SIgA and cortisol levels predict stress-related health outcomes. Stress and Health. 2006; 22: 11–23. 
• Walsh NP, Gleeson M, Shephard RJ, Gleeson M, Woods JA, Bishop NC, Fleshner M, Green C, Pedersen BK, Hoffman-Goetz L, Rogers CJ, Northoff H, Abbasi A, Simon P. Position statement. Part one: Immune function and exercise. Exerc Immunol Rev. 2011;17:6-63. Review.

Use Glutamine to Heal the Gut and Hinder Your Gut Bacteria from Eating Away Your BCAA, Arginine and Other Aminos

Image 1: Don't do this at home. A pig with cannules that have been dug into its digestive tract to study chemical enzymatic and microbial actions (University of Illinois)

With terms like "the leaky gut syndrome" and the usual quick (and not so quick) fixes a la "solutions for a leaky gut" being all over the blogosphere, I suspect that I am not telling you anything new, when I say that there is reasonable scientific evidence that glutamine is good for your gut (nice alliteration, by the way ;-). That it is yet also "good" for the bacteria in your gut may be news to you. According to a recently published study by scientists from the Laboratory of Gastrointestinal Microbiology, at the Nanjing Agricultural University in Nanjing, China, dietary l-glutamine can exert direct regulatory effects on the amino acid utilization of your gut bacteria - specifically their uptake and use of l-arginine and related amino acids (Dai. 2012).

"Hey, you gut bug, leave my arginine alone!"

It has been known forever that l-glutamine is the most abundant amino acid in the body. It's sheer ubiquitousness and rapid turnover relfect its crucial role in whole body nutrient metabolism and health. While fitness and health enthusiasts have long been mislead to believe that glutamine would exert direct ergogenic effects (cf. Amino Acids for SuperHumans: Part IV - Glutamine), scientists and laymen alike are now zoning in on its protective effects on the integrity and function of the small intestine. In this context it has recently become clear that

... AA [amino acid] metabolism in the small intestine plays important roles in the regulation of whole-body AA homeostasis. [...] Recent studies suggest that bacteria in the small intestine are active in the metabolism of AA, especially lysine, threonine, arginine, glutamate and glutamine. The rapid utilization and metabolism of glutamine by small-intestinal bacteria supports the view that glutamine is a key regulator of the survival and growth of bacteria in the intestine through the regulation of the bacterial metabolism of nitrogenous compounds particularly AA.

The usage of glutamine by your gut bacteria is yet not the only physiologically significant interaction between dietary glutamine and the billions of microorganisms in your gastrointestinal tract. According to Dai et al. glutamine may also affect the utilization and metabolism of other amino acids by small-intestinal bacteria "and metabolism in small-intestinal bacteria, and thusly influence the "the production and profile of nitrogenous compounds in the lumen of small intestine and whole-body amino acid homeostasis."

Complex interactions with a simple solution? Ramp up your glutamine intake!?

As you can see in figure 1 the influence of dietary glutamine on the uptake of nitrogen amino acids by Streptococcus sp., Escherichia coli, Klebsiellasp. and a number of jejunal mixed bacteria or ileal mixed bacteria from the small intestine of pigs (as omnivores they have a very similar digestive tract as humans) was profound, yet dose depended and overall pretty complicated.

Figure 1: Change in utilization [nmol /( 10 cells 3h )] of amino acids from the l-arginine family by small intestinal bacteria subsequent to incubation with 0, 0.5, 1, 2 and 5 mmol/L l-glutamine (data adapted from Dai. 2012)

While the incubation of the bacteria with different amounts of l-glutamine lead to increases and decreases of amino acids from the l-arginine family (cf. figure 1), its addition to a medium with the three BCAAs, leucine, isoleucine and valine (cf. figure 2) led to dose-dependent decreases in all three branched-chain amino acids and may thusly explain why direct ergogenic effects of l-glutamine usually occur in the context of low protein diets, only.

Figure 2: Change in utilization [nmol /( 10 cells 3h )] of branched-chain amino acids by small intestinal bacteria subsequent to incubation with 0, 0.5, 1, 2 and 5 mmol/L l-glutamine (data adapted from Dai. 2012)

The addition of glutamine to low protein diets (and or low dose BCAA supplements) may simply facilitates that the small amount of BCAAs that is present in the die / supplement, will actually make it to the gut border and into your system (and is not "eaten by bacteria" before), where it (and leucine in particular) is then able to exert its mTOR-mediated protein synthetic effects.

Species, dosage and amino acid dependence - further investigations necessary

If you take a look at all the data from the Dai study, it is immediately evident that things are way more complicated than a cursory look at the graphs in figure 1-2 would suggest:

• the net effect on the metabolism of amino acids from the arginine-family is particularly pronounced; the production / conversion of arginine to ornithine and citrulline increased and the overall use of arginine by the bacteria decreased (in some cases profoundly) 
• the increased glutamate production from E. coli could be of particular importance, in view of the multi-faceted function of glutamate in the mammalian (and thusly human, as well) metabolism
• the decrease in the utlization of BCAAs, but also the amino acids from the serine aspartate family, i.e. l-alanine, l-asparagine, l-aspartate, glycine, l-serine and l-threonine allow for a greater uptake and utilization of these physiological important amino acids by the enterocytes

All these factors lead the scientists to conclude that high enough amounts of dietary or supplemental "glutamine [are] not only nutritionally important, but also crucial for maintaining gut health and function", because

• 

  Illustration 1: Potential gut microbiome mediated benefits of higher dietary or supplemental l-glutamine intake; maybe it's still worth taking although it's not a proven ergogenic

  they will fulfill the glutamine requirements of the potential pathogenic bacteria E. coli and Klebsiellasp. and thusly make up for their detrimental effect on the amino acid availability of the host
• the thusly well-nurished and healthy gut lining protects the host (you) from bacterial invasion and infection
• with their role in the intestinal signaling pathways, the increased availability of arginine, amino acids from the arginine family and their related metabolites like agmatine and polyamines can exert direct regulatory effects on gut function and integrity
• there is emerging evidence that the degradation of serine and aspartate, which was reduced in the presence of larger amounts of glutamine, is a major factor contributing to the (over-)colonization of the small intestine with bacteria and the production of virulence factors in the intestine

Despite the fact that these results were obviously obtained by scientists interested in the development of effective feeding strategies for foodstock (ruminents with their large amount of bacteria in particular), the notion that higher glutamine intakes may exert (likely) beneficial effects on both amino acid availability, as well as gastrointestinal integrity and overall health appears to confirm the existing anecdotal evidence that glutamine-rich foods and supplements could make an important, if not essential contribution to your gastro-intestinal and metabolic health.