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 ;-)

Ursolic Acid, Leucine or Placebo for mTOR, IGF-1, Irisin, Size & Strength Gains & Fat Loss? Plus: Tart Cherry Powder as Anti-Oxidant Immune Protector for Athletes

Weights are better than pills, but in some cases, they can also work synergistically.

A new month, a new exercise & supplementation research overview for the "SuppVersity Short News". In today's installment I am going to highlight the latest evidence for the beneficial anti-oxidant & immuno-protective effects of tart cherry powder in endurance athletes. I am going to discuss the disappointing results of a comparison between leucine and ursolic acid as mTOR-elevating, IGF-1 receptor modulators in the post-workout window. And I am going to contrast these findings to the results of a non-sponsored chronic resistance training + ursolic acid intervention from a group of Korean scientists.

Read more short news here at the SuppVersity

Obesity Research Upd. Nov. '14

Exercise Res. Upd. Nov '12(1)

Exercise Res. Upd. Nov '12(2)

Nutrition Res. Update Nov. '14

Weight Loss Tricks & More

Reductive Stress, Iron & the Military

• Tart cherry powder as anti-oxidant immuno-protector - Scientists from the Texas A&M University reported at the Eleventh International Society of Sports Nutrition (ISSN) Conference and Expo that the provision of tart cherries, not as whole fruits, but in 480 mg capsules that contained a freeze dried powder (one capsule daily), for 10 days leading up to a half-marathon, the 27 endurance trained or triathlete (21.8±3.9 yr, 15.0±6.0% body fat, 67.4±11.8 kg) men (n=18) and women (n=9) completed in less than one hour lead to a significant reduction of the immune response to exercise. In particular, the statistical analyses of the data revealed a...

  "[...] significant group x time quadratic effect [...] for WBC [white blood cell count] (p=0.034) [and] a trend toward a significant delta value based on group assignment for WBC (p=0.09)" (Goodenough. 2014).

  In spite of the fact that the mitigated immune response following exercise did, as Goodenough et al. point out "correlate with the decreased catabolic response indicated by BUN/Cr ratio and cortisol levels reported in a companion abstract", and notwithstanding the results of a similar study which found that
  

  "acute supplementation with powdered tart cherries over the 7 days leading up to, during, and 2 days after intense resistance exercise helps to minimize post-training perceptions of pain in the most biomechanically loaded regions of the quadriceps muscle group associated with the back squat compared to a placebo" (Levers. 2014),

  the overall benefits are probably negligible for normal trainees. For training junkies and professional athletes, though, the ameliorated stress and immune response, as well as a minor decrease in pain may yet be good reasons to give tart cherry supplements a try.
  

Homebrew blackberry "supplement" inhibits lipid oxidation.

In case you're not into tart cherries, a recent non-sponsored study that does not use a commercial supplement from the Islamic Azad University (Niloofari. 2014) shows that you can also boil 100g blackberries with some water for 10 minutes, then cool them and place them in a sealed containers in order consume 100ml of the black- berry "soup" daily. In said study the consumption of this extract lead to a significant reduction in the producton of lipid oxidation products in obese subjects during a standar- dized resistance training program. Accordingly, we can assume that this "homebrew" supplement will have similar protective effects on the cell membranes as the capped tart cherry extract from the perviously discussed study.

• Ursolic acid or leucine, what's more anabolic on paper? Yes, there is a good reason I underlined the words "on paper". Why? Well, the experiment David Church, Neil Schwarz, Mike Spillane, Sarah McKinley, Tom Andre and Darryn S Willoughby conducted may employ a randomized, cross-over design, but still investigated only the acute effects of 3g leucine (LEU), ursolic acid (UA) or placebo on IGF-1 (a serum regulator of MPS) and the Akt/mTOR pathway.
  
  The 9 apparently healthy, resistance-trained [regular, consistent resistance training (i.e. thrice weekly) for at least 1 year prior to the onset of the study] men between the ages of 18-30 who had volunteered to participate in this study consumed the supplements immediately afer a lower-body resistance exercise that involved 4 sets of 8-10 repetitions at 75-80% 1-RM on the angled leg press and knee extension exercises. A venous blood sample was obtained before, and 0.5, 2, and 6 hr post-exercise, whereas a vastus lateralis muscle biopsy was obtained before and 2 and 6 hr post-exercise. 
  

  

  Figure 1: the results of the ursolic acid vs. leucine comparison are not exactly exciting (Church. 2014).

  The analysis of these blood and muscle samples revealed that there were no differences  observed among the three "supplements" (one of them being the placebo) for serum IGF-1 (p > 0.05), the expression of IGF-receptors or the phosphorilation of Akt, and p70S6K (p > 0.05).
  
  The only significant difference the researchers observed was a significant increase in phosphorylated mTOR in response to the 3g of leucine compared to UA and PLC (p = 0.001).
   
• Ursolic acid: Different study different outcome - In the defense of ursolic acid it should yet be mentioned that Hyun Seok Bang et al. recently found that the chronic supplementation with 3x450mg/day of ursolic acid lead to concomittant increases in serum irisin and muscle strength, as well as impressive reductions in bod fat in twenty-four Korean men with over 3 years of resistance training experience.
  

  

  Figure 2: Changes in body composition (left) and IGF-1 & irisin levels (right) in response to 8 weeks of resistance training + placebo or resistance training + 3x450mg/day of ursolic acid (Bang. 2014).

  As you can see in Figure 2, significant increases in IGF-1 weren't observed in this study either. Accordingly, Church et al. may simply have been betting on the "wrong anabolic horse" and the chronic effects like the strength gain and fat loss Bang et al. observed in their trained subjects alluded them due to the nature of study, i.e. an acute vs. chronic exercise supplementation regimen.

BCAAs Don't Build Muscle, Study Says | Learn more

Bottom line: In view of the fact that the ursolic acid studies teach us not to judge a supplement by an acute / short-term study I refuse to make a commendation for or against tart cherries - no matter, whether they're fresh or powdered.

What I am willing to admit, though, is that the study by Bang et al. (2014) yielded pretty impressive results. Results that are not yet enough to fully convince me that all the hilarious claims you will find on ursolic acid supplements are true, but results that will have me keep an eye on future studies investigating the effect of chronic ursolic acid supplementation on body composition and physical performance | Comment on Facebook!

References:

• Bang, Hyun Seok, et al. "Ursolic Acid-Induced Elevation of Serum Irisin Augments Muscle Strength During Resistance Training in Men." The Korean Journal of Physiology & Pharmacology 18.5 (2014): 441-446.
• Church, David, et al. "A comparison of the effects of ursolic acid and l-leucine supplementation on IGF-1 receptor and AKT-mTOR signaling in response to resistance exercise in trained men." Journal of the International Society of Sports Nutrition 11.Suppl 1 (2014): P19.
• Goodenough, C., et al. "Powdered tart cherry supplementation mitigates the post-exercise immune response with reduction in total antioxidant status and serum triglyceride levels following an acute bout of intense endurance exercise." Journal of the International Society of Sports Nutrition 11.Suppl 1 (2014): P34. 
• Levers, K., et al. "Powdered tart cherry supplementation demonstrates benefit on markers of catabolism and muscle soreness following an acute bout of intense lower body resistance exercise." Journal of the International Society of Sports Nutrition 11.Suppl 1 (2014): P31. 
• Niloofari, A., Et Al. "Responses Of Oxidative Stress Indices To Resistance Exercise After Blackberry Extract Supplementation." IJBPAS 3.12 (2014): 2798-2810.

Branched Chain Amino Magic: Study Takes Another Step Towards a Better Understanding of the Anabolic & Anticatabolic Effects of BCAAs and Their Essential Cousins

Image 1: Without the other essential amino acids (EAAs), the branched chain amino acids, leucine, isoleucine and valine (BCAAs) have nothing to "build" your muscle from ;-)

Usually, I do not get very excited, when I hit upon another study into the "protein-synthetic response" that is triggered by the ingestion of branched chain amino acids (BCAAs). I mean, let's be honest... we all know that their ingestion will trigger the phosphorylation of the mammalian target of rapamycin and thusly increase protein synthesis, so why would we need another study where instead of a 17.5% increase in protein synthesis, we would see a 18.3% increase? Actually, we don't... the data Marcus Borgenvik, William Apró and Eva Blomstrand from the  Åstrand Laboratory, Swedish School of Sport and Health Sciences and the Karolinska Institutet, in Stockholm, Sweden (Borgenvik. 2011), collected goes yet well beyond what we have seen in most of the previous studies and is thus well worth an individual blogpost here at the SuppVersity.

BCCAs work! How? Little do we know...

If we are honest, we must concede that our (=the scientific) understanding of the complex processes that are triggered when "large" amounts of BCAAs hit our bloodstream, is very limited. What we know is that we can measure increases in mTOR-expression that correlate with likewise measurable increases in protein synthesis. What we do not really know is how exactly one leads to the other and where the influences of amino acid supplementation and exercise training overlap. This is even more true for the complementary side of the protein synthetic equation of which Borgenvik et al. state that

[w]hereas extensive evidence for the stimulatory effect of amino acids, either alone or in combination with exercise, on protein synthesis has been reported, their effect on protein breakdown is elusive.

In that, it is particularly confusing that "previous investigations involving ingestion of essential amino acids (EAA) in connection with resistance exercise have revealed no attenuating effect on protein breakdown", while studies which investigated the effect of BCAA or leucine in isolation, report reduction in protein degradation in subjects at rest or performing eccentric endurance exercise (MacLean. 1994). Reason enough for the Swedish scientists to recruit a group of seven healthy, recreationally active participants (5 men, 2 women; 27 (± 2) years; height 175 (± 5) cm; weight 67 (± 7) kg), put them on a standardized diet (17% protein; 25% fat; 57% carbs; ~2100kcal for women, ~2700kcal for men) for two days and, on the subsequent morning (subjects reported to the lab fasted) and after a thorough warm-up, have them perform

• 4 sets of 10 repetitions at 80% of their predetermined 1 RM, followed by another 
• 4 sets of 15 repetitions at 65% of their 1 RM of single-legged leg presses.

The subjects used the same leg on all exercises and rested ~5min after each set. Before the warm-up, during and immediately after and 15 and 45min after the exercise regimen the subjects consumed either

• 150 mL of BCAAs (2:1:1 ratio) in flavored water, or
• 150 mL flavored water alone.

The total amount of BCAAs was 85mg/kg or 5.695g for the "average" study participant. After four weeks the experiment was repeated with each participant receiving the opposite treatment.

Figure 1: Complete analysis of serum amino acid levels in the trained and untrained leg of subjects receiving BCAA or Placebo supplement before, during and after the completion of a standardized single-legged leg press exercise (data adapted from Borgenvik. 2011)

As far as the study protocol goes this is thus certainly not an extraordinary study. If you take a look at figure 1, where I deliberately plotted all the data the scientists gathered as far as serum amino acid concentrations are concerned, you will yet realize that what makes this study stand out is the sheer amount of parameters Borgenik et al. have analyzed. Similar data is also available for the amino acid concentrations in the exercised muscle and though, the scientists, who set out to investigate the effects of BCAA supplementation on protein breakdown, would probably disagree with me, here, I feel that this data actually has the most real world significance for physical culturists, like you and me.

Figure 2: Relative increase / decrease in intra-muscular BCAAs and other EAAs in BCAA supplemented subjects vs. placebo control at different time-points before, during and after single-legged leg presses (data adapted from Borgenvik. 2011)

After all, a brief glance at the effects the ingestion of ~6g of BCAA had on the respective tissue levels of leucine, isoleucine and valine (figure 2, BCAA) and the other, "missing" essental amino acids (figure 2, EAA - BCAA) should suffice to understand that though BCAAs may be the necessary to trigger protein synthesis, they are yet obviously not sufficient to "build muscle" - or how else would you explain the

pronounced reduction in the concentration of the aromatic amino acids, tyrosine and phenylalanine, in both plasma and muscle as well as muscle EAA (BCAA excluded) during the recovery period

Borgenik et al. observed in their study? The scientists at least conclude that

[s]ince tyrosine and phenylalanine are neither synthesized nor degraded in skeletal muscle, reduction in the levels of these amino acid could be indicative of an improved net muscle protein balance, i.e. an enhanced rate of synthesis and/or decreased rate of breakdown [and] could  be explained by incorporation into protein.

The accrual of muscle mass (whatever that may eventually mean, cf. yesterday's installment of the Intermittent Thoughts) thusly obviously relies on the presence of all essential amino acids and not just the "branched chained holy grail" of protein synthesis, of which the current study revealed that they (BCAA ingestion) reduced the expression of MAFbx, which regulates the protein transcription factor MyoD and the eukaryotic initiation factor-3f (eIF-3f), which, in turn is of importance in the mTOR-p70S6k signaling pathway, by 30% and 50% in the resting and exercising legs, respectively.

Figure 3: Relative (compared to placebo) mTOR and p70S6K phosphorylation in response to BCAA supplementation in exercised (EX) and non-exercised (Rest) leg at different time-points before, and after single-legged leg presses (data adapted from Borgenvik. 2011)

As figure 3 finally goes to show, we see the "usual" increases in mTOR and p70S6K phosphorylation that are commonly held responsible for the downstream increases in protein synthesis, and which were obviously more pronounced in the exercised compared to the non-exercised leg. The latter may be ascribed to what the scientists cautiously label a ...

[...] tendency for BCAA supplementation to attenuate the elevation in the level of Rheb mRNA in both resting (1.7-fold under the placebo versus 1.2-fold in the BCAA condition) and exercising muscle (2.4-fold versus 1.5-fold).

This ameliorative effect on Rheb, the low-molecular weight GTPase located immediately up-stream of mTOR, in combination with the exercise induced reductions in REDD2 expression (another negative regulator of mTOR) the scientists observed in the exercised leg are actually where we are currently at, as far as our understanding of the complex protein synthetic machinery goes. It is here at the gene-level where amino acid supplementation and its effect on Rheb and exercise and its effect on REDD synergize and facilitate those muscle gains trainees have been making for years often without any understanding of the biological underpinnings.

And though we may eventually be able to squeeze out another 5-10% more muscle mass, when we eventually get the "whole picture", I seriously doubt that even the most thorough understanding of the underlying biomolecular processes will change such basic recommendations as "take your 25g of fast digesting whey as a bolus immediately post workout" (cf. "Never Sip Your Whey!") - or what would you say?

Whey Protein Alone Won't Cover the EAA Requirements of Hard Working Athletes, Study Says. Plus: US Whey More Digestible & 88% Higher in Leucine than Brazilian Whey

Not all protein supplements are created equal. And this goes for whey supplements from different countries, too.

In their accepted manuscript for LWT - Food Science and Technology, Cristine Couto Almeida and her colleagues write: "When the calculated AAS and PDCAAS based on the suggestion for adult athletes were considered, both [US & Brazilian whey protein] supplements exhibited suboptimal score values for several EAA [... and] were unable to supply the suggested adult athlete EAA requirement" (Almeida. 2014).

Shocked? I'd hope not. I mean, you don't even know what the scientists base their conclusion on - right? So before we even try to put things into perspective, it would be wise to take a look a the design of this in vitro study.

You can learn more about protein intake at the SuppVersity

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While the researchers from the Universidade Federal de Rio de Jaieiro acknowledge that whey protein, in general, is an effective adjunct to the diet of strength and even endurance athletes, they insist that there is too little "information regarding the WP supplement protein quality" and thus set out to "to investigate the protein quality of commercial WP supplements produced by U.S. and Brazilian companies based on in vitro digestibility (IVPD) assay, EAA, AAS and [protein digestibility-corrected amino acid] PDCAAS." (Almeida. 2014)

To this ends, the researchers acquired fifteen samples of whey protein (WP), soy protein, and caseinate isolate powder from a commercial retailer specialized on nutritional supplements. The supplements had been manufactured at different countries - eight from USA companies (WP-USA), and seven from Brazilian companies (WP-BRA). The supplements manufactured with soy protein and caseinate isolate powder were used as references in a study that yielded quite surprising results.

Figure 1: Essential amino acid composition of two commercial whey protein supplements (Almeida. 2014).

As you can see in Figure 1 the amino acid composition of the whey proteins from Brazil and the US varied significantly. The US whey, for example had significantly higher amounts of leucine, while the Brazilian whey was loaded with the essential amino acid lysine. While it is possible that the variations in the other amino acids are a result differences that were present in the milk, already, I would guess that the US whey was either openly (the scientists don't disclose the brands, otherwise I'd check) or secretly spiked with leucine to promote muscle anabolism.

Figure 2: Relative loss (%) of amino acids during simulated (in vitro) digestion in US and Brazilian whey (Almeida. 2014).

What are the numbers based on: Whether the amount of aminos is sufficient or not was calculated based on the WHO recommendation (WHO. 2007), assuming a normal (=comparatively low) protein intake.

If you consume twice the WHO suggestions for athletes, you are thus not at a risk of being deficient in any of the EAAs, but could maybe optimize the ratio of the individual amino acids by not covering your protein needs from a single protein source.

Even if we assume the latter was the case and the producer added free form amino acids to the whey, though, this does not explain the other differences, because if you add say 20g of leucine to 100g of EAA and measure the amino acid content of the 100g of your new mix, the content of all other amino acids would be lower.

 As you can easily see in Figure 1, though, this was not the case in the study at hand. Plus: There were also significant differences in the protein digestibility-corrected amino acid composition, i.e. the marker of whether or not the content of a certain essential amino acid per gram of protein was sufficient or not. In that, values <1.0 indicate there is too little of this amino acid in the mix.

Figure 3: Amino acid score and protein digestibility-corrected amino acid composition for the commercial
US and Brazilian whey supplements (Almeida. 2014).

As you can see in Figure 3, the latter was the case for threonine and valine in the US whey and for isoleucine and leucine in the whey protein from Brazil.

Figure 4: According to the standardized in vitro digestion assay (AOAC. 2012) the scientists used soy protein has by far the lowest digestibility and will thus be effectively delivering the lowest percentage of the amino acids it contains into your circulation (Almeida. 2014).

What does this mean? I must admit this sounds awful, but in practice it means only that you would end up getting your EAAs at an allegedly suboptimal ratio (I doubt we know what this ratio is, though) if you covered your complete protein needs with whey protein. In that, it is interesting that you would get too little threonine and valine form US wheys and too little leucine and isoleucine from Brazilian wheys.

Actual deficiency symptoms as you may have expected them, when you've read the statement that whey protein supplements were "unable to supply the suggested adult athlete EAA requirement" (Almeida. 2014), however are unlikely, because (a) I assume most of you won't live off whey protein as their only protein source and (b) even if you did, you would probably consume more than the WHO recommendation for athletes (WHO. 2014) that's at the heart of Almeida et al.'s calculation suggests | Comment of Facebook!

References:

• Almeida, Cristine Couto, et al. "In vitro digestibility of commercial whey protein supplements." LWT-Food Science and Technology (2014). 
• AOAC International, and George W. Latimer. Official Methods of analysis of AOAC International. AOAC International, 2012.
• Hsu, H. W., et al. "A multienzyme technique for estimating protein digestibility." Journal of Food Science 42.5 (1977): 1269-1273.
• WHO. "Protein and amino acid requirements in human nutrition." World Health Organization technical report series 935 (2007):

The Counterintuitive Catabolic & Pro-Diabetic Effects of Leucine Supplementation in Rodents on Corticosteroids

Not the mice from this study, but still a nice example of the effects of dexamethasone on skeletal muscle (right; Quin. 2012)

"Leucine-laced water + stress = insulin resistance" - This simple equation is the net result of a recent study by Nelo Eidy Zanchi and his colleagues from the Laboratory of Applied Nutrition and Metabolism at the School of Physical Education and Sports of the University of Sao Paulo in Brazil. Inspired by previous research which clearly indicated that leucine does not only have pro-anabolic, but also insulin sensitizing effects, Zanchi et al. speculated that the provision of adequate amounts of leucine would blunt the catabolic and pro-diabetic effects of 7 days of intraperitoneally injections of  dexamethasone, an artificial corticosteroid that's used to treat all sorts of inflammatory diseases.

Remember SuppVersity Rule of Smart Supplementation No. 2? Right. Specificity!

In order to test their hypothesis that leucine supplementation either in low doses in the drinking water or as higher dosed oral gavage would ameliorate the negative side effects of DEXA treatment, the scientists randomized groups of 10 male Wistar rats to six groups receiving either low dose or high dose leucine supplements with and without dexmethasone.

"During  the duration of the experiment, which lasted  seven  days,  DEXA (a synthetic glucocorticoid analogue that does not bind to plasma binding proteins) was given daily (at 9:00 a.m.) through intraperitoneal injection (5 mg/kg/day); control groups received  an equivalent volume of saline (0.9% NaCl). As DEXA was reported to decrease food intake, all groups were  fed the same amount of food (in terms of caloric intake) equal to the DEX group. Thus, differences among groups did not originate from different food intakes. We measured the caloric content of our standard chow (16.32 kJ/g) as well as leucine (25 kJ/g) in a calorimetric bomb (FTT Oxygen Bomb Calorimeter) in  order to avoid differences in the caloric ingestion between experimental groups and observed that the total caloric consumption was not statistically different among groups." (Zanchi. 2012)

The leucine was administered either in dosages of 0.068g/kg body weight per day (low dose) or 1.35 g/kg per day (high-dose) twice daily at 8:00  a.m. and  2:00  p.m. through gavage over seven days. And while the scientists had selected the high dose (LH) "to induce a maximal increase in muscle protein synthesis and insulin plasmatic levels", the dosage in the LL (=low leucine) group was too low to increase either muscle protein synthesis or plasma insulin levels. The third, non-supplemented control group received an NaCl (sodium) placebo, the volume of which was identical to the supplement to make sure that any possible volume-induced effects of oral gavage that could for example be induced by gastric expansion would not skew the study results.

"But leucine has been shown to be anabolic! So it must help."

Aside from the usual basal fasting glucose, insulin, tryacilglycerol (TAG) and HOMA-IR values, the scientists did also assess the motor performance of the animals by the means of two standardized strength and ambulation tests (Kennel. 1996; Anderson. 2004; Viera. 2008).

Figure 1: Effect of 7 days of low (LL) and high (LH) dose leucine supplementation with and with out dexamethasone on total body mass, soleus (slow twitch) and EDL (fast twitch) muscle mass in male Wistar rats (left; values expressed relative to non-supplemented control) and corresponding changes in mean ambulation and grip strength (right; Zanchi. 2012)

As you can see in figure 1 the supplemental leucine failed to reduce the negative side effects of dexamethasone. As far as the total body weight and the fast-twitch muscle mass (EDL) are concerned, you could even argue that the high dose treatment (DEX-LH) did even amplify the catabolic effects of the synthetic corticosteroid:

"Thus, leucine supplementation at both low and high doses did not counteract body weight loss in both food restricted (control groups) and DEXA-treated animals. Soleus muscle mass did not differ among groups. Leucine supplementation at  high doses  attenuated food  restriction-induced EDL muscle loss (CON-LH group) when compared with the CON-NS group  (p < 0.05). All DEXA-treated animals presented reduced EDL muscle mass when compared with the CON-NS group (p < 0.05), and leucine supplementation at both low and high doses of amino acid did not attenuate it." (Zanchi. 2012)

Now, you may well argue that the mere fact that the muscle weight was "statistically significant" reduced, this does not mean that these reductions would be physiologically significant and that the minimal differences between the DEX groups would not matter, anyway. If you just go by the data on the left side of figure 1, this is certainly right, if you do yet also consider the significant reductions in muscle function (figure 1, right) and the fact that all that happened within no more than 7 days, the overall result should actually remind you of the "Three Simple Rules of Smart Supplementation" - and here specifically the 2nd one: Specificity!

Figure 2: Time course of the dexamethasone-induced detoriations in fed serum glucose levels and ameliorative effect of low and high dose leucine supplementation (left) and effects of the treatment on fasting insulin levels and HOMA-IR (index of insulin resistance) at the end of the study (Zanchi. 2012)

In fact, the data in figure 2 only confirms the notion that things that you cannot define "good and bad", "black and white" and "beneficial or detrimental" without a context and the outcome you are expecting. If you are trying to keep the postprandial blood sugar in check, for example he addition of an effective (high dose) of leucine to the diet would appear to be a good idea. If, on the other hand, you are more concerned about insulin resistance, you would be better advised to use minimal amounts of leucine or simply refrain from supplementation altogether.

Figure 3: If the ingestion of bolus amounts of leucine is not helpful, lacing the water of the rodents DEXA treated rodents with leucine turned them into full-blown diabetics (Zanchi. 2012)

As these results clearly demonstrate the provision of additional leucine is not useful to counter the negative side-effects of synthetic corticosteroids. On the contrary, the negative effects on insulin resistance are apparently even augmented and the muscle function is further compromised by the purpotedly anabolic high dose leucine supplement.

And while the overall effects of the bolus administration may still be negligible, the scientists ingenious idea that the provision of similar amounts of leucine in the drinking water in a second follow-up experiment turned out to be "capable of inducing a massive diabetic state" (Zanchi. 2012; see figure 3 for the ensuing surge in fasting blood glucose levels) while decreasing the mass of the fast-twich EDL muscles even further.

Bottom line: Overall these results only confirm the simple, but often neglected truth that inductive reasoning is a futile undertaking in the realms of exercise and nutrition sciences: What is good for an athlete is rarely optimal for an obese person, the same diet that helps the obese lose weight, will make the athlete feel miserable, and lacing the drinking water of rodents on corticosteroids with the exact same amount of leucine that has had highly beneficial effects on the insulin sensitivity of diabetic rodents in previous studies (Guo. 2010) will not only fail to ameliorate the glucocorticoid-induced detoriations in blood glucose, it will even exasperate them.

So, does that mean you should not take your whey protein or BCAAs any longer? No, if you did that you would make the exact same mistake as someone who laces his water with leucine in order to avoid the catabolic effects of the synthetic corticosteroid he is taking for medical reasons. On the other hand, the results of the study at hand should make you re-evaluate the necessity and even benefits of guzzling BCAAs all-day long, at least if the reason for doing so is that you believe that you are so stressed that you would otherwise fall into a catabolic black hole.

That said, there may even be implications for the average pre-diabetic inhabitant of the Western hemisphere who is eating his hamburger and French fries on the parking lot of the local fast food restaurant, because he cannot make room to prepare and consume a real meal somewhere in his busy and stressful schedule. I mean, despite the fact that the aforementioned specificity principle does not allow for anything but a still to be verified hypothesis, it does at least appear not to far-fetched that this chronic endogenous stress, despite being very different from the "stress" that's induced by the administration of a synthetic corticosteroid that does not bind to serum proteins, could have similar negative modulatory effects on the purported benefits of chronic leucine supplementation ... but as I've said before, this would be something to investigate in another study. So unless you are actually taking dexamethasone for medical reasons, you are probably not at risk of developing diabetes due to a high amount of leucine in your diet.

In the unfortunate case that you are actually on synthetic corticosteroids, a previous study by the same group of scientists, in the same rodent model does suggests that three workouts with three sets of squats (10 reps each) per week may offer the protection against corticosteroid induced muscle loss decreased skeletal muscle GLUT-4 expression and insulin resistance, leucine does not have to offer.... well, at least as long as you abstain from leucine supplementation, because the latter had the exact same detrimental effects in the 2011 study where it was administered to one of the experimental groups in conjunction with resistance training as it had in these more recent experiments in the absence of any type of workout (Nicastro. 2011). 

References:

• Anderson,  K.D.; Abdul, M.; Steward, O. Quantitative assessment of deficits and recovery of
  forelimb motor function after cervical spinal cord injury in mice.  Exp. Neurol.  2004,  190,
  184–191.
• Kennel,  P.F.; Fonteneau, P.; Martin, E.;  Schmidt,  J.M.; Azzouz, M.; Borg, J.; Guenet,  J.L.;
  Schmalbruch, H.; Warter, J.M.; Poindron, P. Electromyographical and motor performance studies
  in the pmn mouse model of neurodegenerative disease. Neurobiol. Dis. 1996, 3, 137–147.
• Nicastro H, Zanchi NE, da Luz CR, de Moraes WM, Ramona P, de Siqueira Filho MA, Chaves DF, Medeiros A, Brum PC, Dardevet D, Lancha AH Jr. Effects of leucine supplementation and resistance exercise on dexamethasone-induced muscle atrophy and insulin resistance in rats. Nutrition. 2012 Apr;28(4):465-71. Epub 2011 Nov 12.
• Qin J, Du R, Yang YQ, Zhang HQ, Li Q, Liu L, Guan H, Hou J, An XR. Dexamethasone-induced skeletal muscle atrophy was associated with upregulation of myostatin promoter activity. Res Vet Sci. 2012 Aug 29.
• Vieira, N.M.; Bueno,  C.R., Jr.; Brandalise, V.; Moraes,  L.V.; Zucconi, E.; Secco, M.; Suzuki, M.F.; Camargo, M.M.; Bartolini, P.; Brum, P.C.; Vainzof, M.; Zatz, M. SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression.  Stem Cells  2008,  26, 2391–2398.  
• Zanchi NE, Guimarães-Ferreira L, de Siqueira-Filho MA, Felitti V, Nicastro H, Bueno C, Jr, Lira FS, Naimo MA, Campos-Ferraz P, Nunes MT, Seelaender M, de Oliveira Carvalho CR, Blachier F, Lancha AH, Jr. Dose and Latency Effects of Leucine Supplementation in Modulating Glucose Homeostasis: Opposite Effects in Healthy and Glucocorticoid-Induced Insulin-Resistance States. Nutrients. 2012; 4(12):1851-1867.

Chronic High Dose BCAA Supplementation Reduces Endurance Performance by 43% Plus: How Ammonia, Glutamine, Arginine & Low Carb Could be Involved

Tired, exhausted, had to cut your workout short today? Is it the flu, or just too much BCAAs?

When some is good and more is better, even more is not necessarily going to be 'betterer' - and that's not simply due to the fact that there is no comparative to an adjective that's already in the comparative. Therefore it is actually not surprising that a team of researchers from the Department of Food and Experimental Nutrition at the Faculty of Pharmaceutical Sciences, the Department of Nutrition at the School of Public Health and the Department of Physiology and Biophysics at the Institute of Biomedical Sciences of the University of Sã o Paulo in Brazil has just published the results of a study (Falavigna. 2012) which demonstrates that there is an upper limit to the benefits of BCAA supplementation. What I guess will be surprising at least for some not so regular SuppVersity visitors, is that there is more than just a saturation effect: Too much BCAAs can actually have ergolytic (= anti-ergogenic) effects - at least under certain circumstances.

Another chapter in the book of good things that turn against you, when taken in excess

In their latest paper that has just been published in nutrients, Gina Falavigna and her colleagues analyzed effects of chronic BCAA supplementation on exercise performance in male Wistar rats. Based on previous animal and human data and the still widely supported, though actually experimentally non-validated (cf. Meeusen. 2007) theory that BCAAs would work their non-hypertrophy specific, endurance enhancing magic via the blockade of exercise induced 5-HT (serotonin) accumulation in the brain, the researchers speculated that ...

"[...] chronic BCAA supplementation (through the diet, using different BCAA  concentrations) would increase performance in rats subjected to a swimming exhaustion  test." (Falavigna. 2012)

To verify this hypothesis, Flavigna et al. randomized their rats to three different groups receiving either the standard AIN-93M diet for the maintenance of adult rodents (control group) or the same diet with additional additional 3.57% (group S1) and 4.76% (group S2) BCAAs at a ~2:1:1 ratio of lecine : valine : isoleucine (the BCAAs were manufactured by the Brazilian branch of Ajinomoto). The rodents in the S1 and S2 groups did thus receive 50% and 100% more branched-chain amino acids than the rodents in the control group which had to contend themselves with the BCAAs in the casein fraction of their diets (see figure 1, right). In order to assure that the diets would be isocaloric, an amount of starch equivalent to the amoung of BCCAs that had been added to the chow was removed from the supplemented diets.

Overall, the study lasted for six weeks. During this time the rodents were subjected to a 1h/day weight bearing swimming protocol five times a week. In the first two weeks, the rats were ...

"[...] adapted to the water medium and exercised with increasing overloads attached to the tail until an overload corresponding to 5% of total body weight was reached. This final overload was used until the end of the training protocol [...] The overloads were corrected weekly according to the variations in animal weight.  The efficiency of the training protocol was assessed on the basis of maximum activity of the enzyme citrate synthase in the soleus muscle, with a group of sedentary animals being used as the control for this parameter." (Falavigna. 2012)

Neither the overall amount of food nor the body weight gain of the rodents in the control, and the two exercise groups showed any statistically significant difference. The latter cannot be said about the exercise performance, as well as the accumulation of ammonia, though (see figure 1):

Figure 1: Exercise duration and plasma ammonia levels during / after swmming test (left) and macronutrient composition of the experimental diets (right; based on Falavigna.. 2012)

While the rodents in the +50% BCAA group (S1) do show the expected increase in endurance (+37%) their peers in the high dose (+100%) BCAA group (S2) experienced an even more pronounced drop in endurance performance (-43% vs. control), which went hand in hand with a profound increase in blood ammonia (+34%).

"Ammonia is a ubiquitous metabolic product producing multiple effects on physiological and biochemical systems. Its concentration in several body compartments is elevated during exercise, predominantly by the increased activity of the purine nucleotide cycle in skeletal muscle. Depending on the intensity and duration of exercise, muscle ammonia may be elevated to the extent that it leaks (diffuses) from muscle to blood, and thereby can be carried to other organs. The direction of movement of ammonia or the ammonium ion is dependent on concentration and pH gradients between tissues. As such, ammonia can also cross the blood-brain barrier, although the rate of diffusion of ammonia from blood to brain during exercise is unknown. It seems reasonable to assume that exhaustive exercise may induce a state of acute ammonia toxicity which, although transient and reversible relative to disease states, may be severe enough in critical regions of the central nervous system (CNS) to affect continuing coordinated activity. Regional differences in brain ammonia content, detoxification capacity, and specific sensitivity may account for the variability of precipitating factors and latency of response in CNS-mediated dysfunction arising from an exercise" stimulus, e.g., motor incoordination, ataxia and stupor. There have been numerous suggestions that elevated ammonia is associated with, or perhaps is responsible for, exercise fatigue, although evidence for this relies extensively on temporal relationships." (Falvigna. 2012; my emphasis)

Mark the last words of the previously cited paragraph: "[E]vidence for [the role of ammonia] in exercise fatigue relies extensively on temporal relationships". It is thus - as for now - a solely corollary, not yet a causative association, of which I do however feel that it would be very likely to turn into a causal one if someone actually measured the influx of ammonia into the brain during a workout.

Wait, ammonia? But ain't it more likely that the BCAAs block the uptake of tryptophan?

What's for sure is that another hypothesis, which relates to the blockade of tryptophan uptake can be ruled out as an underlying reason of the differences. After all the scientists who argue that ...

"[t]he increased synthesis of serotonin during exercise may be related to the development of central fatigue, because this neurotransmitter has several physiological functions, since it operates by  mood, lethargy, individual behavior, regulation of sleep, body temperature and blood  pressure, appetite suppression and changes in perceived exertion." (Falavigna. 2012)

...actually measured the 5-HT levels and observed no differences between the dietary groups. Overall, the study results to thus clearly indicate that both, medium nor high dose "chronic BCAA supplementation was not effective in improving the main parameters indicative of central fatigue" (Falavigna. 2012) - well, at least as long as we still stick to the hypothesis that the latter is induced by the accumulation of 5-HT in the brain.

Forget about tryptophan and serotonin, focus on ammonia

The fact that neither the high, nor medium dose of BCAAs did exert any effects on the serotonin levels in the brain does yet not explain why the medium dose supplementation regimen produced ergogenic, while the high dose regimen induced ergolytic effects.

The occurrence of direct toxic effects due to (too) high amounts of branched-chain amino acids can be ruled out based on previous studies in which the administration of more than 10g/kg body weight of BCAAs (the human equivalent would be 130g+ per day), as well as dosages of 2.5g/kg body weight chronically did not entail any toxic side effects (Shimomura.  2004). The same is true for other confounding variables, such as the citrate synthase activity, a measure of the general efficiency of the training protocol, bood glucose, insulin,free fatty acids, and lactate levels, as well as liver and muscle glycogen content, which were virtually identical in both groups. This leaves us with the increase in plasma ammonia as our 'last resort' to explain the -58% shorter swimming time in the high (S2) vs. medium (S1) dose BCAA group (-43% lower vs. non-supplemented control).

Figure 2: The reduced performance of the high BCAA group could well be related to peripheral and/or central ammonia build-up as a results of increased BCAA oxidation, camparably low glutamine intakes and the rate-limited enzymantic conversion and recycling of gluatmine (illustration originally from Earrante. 2003). Studies by Snow (2000) and Carvalho-Peixoto (2007) suggest: Both carbohydrate & glutamine supplements could help.

Based on what we know about the mammalian body, the increased build-up of ammonia in the high BCAA group could be a result of the unfortunate combination of temporary energy shortage and learned wastefulness' in a situation, where the otherwise sparse BCAAs are available in abundance. Furthermore, with a glutamine content of only 9-13% in the casein fraction of their diets (Swails. 1992), the rodents in the high BCAA group did ingest more than 2.6-3.8 times more BCAAs than glutamine; a fact which may have contributed to a temporary glutamine deficiency as a result of its increased use in the detoxification of the ammonia that's generated when the BCAAs are oxidized. The resulting peripheral and possibly central ammonia build-up (see figure 2) could then have begun to intoxicate liver and brains of the rodents and thus hampered gluconeogensis (normal levels stimulate, high levels of ammonia hamper gluconeogensis; cf. Fritz. 1988) and induced central fatigue (Wagenmakers. 1990; Nybo. 2004) -- and that not despite, but rather due to the chronic "high dose" BCAA supplementation (HED ~50g/day).

So do I have to drop my BCAAs now or what? Whether these results are relevant for you will probably depend on a whole host of parameters, which include

• the type, intensity and duration of exercise you do, 
• the ratio of BCAAs to glutamine in your diet,
• the amount of arginine, which acts as a substrate for the urea cycle and is therefore necessary to for the excretion of ammonia by the kindeys (Schaefer. 2002),
• the amount of carbohydrates in your diet (with more = less amino acid oxidation = lower ammonia and very low carb = you are in trouble; e.g. Czarnowski. 1995; Snow. 2000; Carvalho-Peixoto. 2007), 

... and those factors I will probably have forgotten to mention now. Unless you don't forget that you can neither lifve from BCCAs and protein alone, but accept the neflglected truth that too much protein is about as bad a too little protein, you can file this post under "show your stupid friends" and get back out, when they complain about feeling sick, bloated and fat "despite" eating a BCAA supplemented high protein, low carb (and often even low fat) diets.

References:

• Carvalho-Peixoto J, Alves RC, Cameron LC. Glutamine and carbohydrate supplements reduce ammonemia increase during endurance field exercise. Appl Physiol Nutr Metab. 2007 Dec;32(6):1186-90.
• Errante LD, Petroff OA. Acute effects of gabapentin and pregabalin on rat forebrain cellular GABA, glutamate, and glutamine concentrations. Seizure. 2003 Jul;12(5):300-6.
• Falavigna G, de Araú jo Junior JA, Rogero MM, de Oliveira Pires IS, rio Graç a Pedrosa R, Martins Junior E, Alves de Castro I, Tirapegui J. Effects of Diets Supplemented with Branched-Chain Amino Acids on the Performance and Fatigue Mechanisms of Rats Submitted to Prolonged Physical Exercise. Nutrients 2012. 4; 1767-1780.
• Fritz S, Bohnensack R. Stimulation of alanine metabolism in rat liver by ammonia. Biomed Biochim Acta. 1988;47(12):923-32.
• Meeusen R, Watson P. Amino acids and the brain: do they play a role in "central fatigue"? Int J Sport Nutr Exerc Metab. 2007 Aug;17 Suppl:S37-46.
• Nybo L, Dalsgaard MK, Steensberg A, Møller K, Secher NH. Cerebral ammonia uptake and accumulation during prolonged exercise in humans. J Physiol. 2005 Feb 15;563(Pt 1):285-90. Epub 2004 Dec 20. 
• Schaefer A, Piquard F, Geny B, Doutreleau S, Lampert E, Mettauer B, Lonsdorfer J. L-arginine reduces exercise-induced increase in plasma lactate and ammonia. Int J Sports Med. 2002 Aug;23(6):403-7.
• Shimomura, Y.; Murakami, T.; Nakai, N.; Nagasaki, M.; Harris, R.A. Exercise promotes BCAA catabolism:  Effects  of BCAA supplementation on skeletal muscle during exercise.  J. Nutr.  2004, 134, 1583S–1587S.
• Snow RJ, Carey MF, Stathis CG, Febbraio MA, Hargreaves M. Effect of carbohydrate ingestion on ammonia metabolism during exercise in humans. J Appl Physiol. 2000 May;88(5):1576-80.
• Swails WS, Bell SJ, Borlase BC, Forse RA, Blackburn GL. Glutamine content of whole proteins: implications for enteral formulas. Nutr Clin Pract. 1992 Apr;7(2):77-80.
• Wagenmakers AJ, Coakley JH, Edwards RH. Metabolism of branched-chain amino acids and ammonia during exercise: clues from McArdle's disease. Int J Sports Med. 1990 May;11 Suppl 2:S101-13.

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

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

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

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

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

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

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

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

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

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

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

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

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

Leucine Only Tops Ergogenic Effects of BCAAs: Increased Alanine Cycle Activity Spares Muscle Glycogen, Boosts Endurance Performance - BCAAs Have Opposite Effect

Alanine is the liver's favorite gluconeogenic amino acid and leucine appears to increase its usage.

Being among the first to learn about the "Glucose-Repartitioning Effect of Iso-Leucine" in February 2013 (read up on it), you, as SuppVersity reader, belong to the selected few who know that valine and isoleucine may be more than unnecessary props in the leucine-powered BCAA show. With the recent publication of a rodent study from the University of Sao Paulo in Brazil (Campos-Ferraz. 2013), however, it looks as if you had to revise your perspective on the purportedly auxiliary BCAAs - at least, with respect to their ability to reduce fatigue, and muscle and liver-glycogen degradation, in trained rats and possibly (!) humans.

So what did the Brazilian researchers do?

Basically, the idea Campos-Ferraz et al. had in mind, when they came up with their 8 week exercise + 2 week supplementation protocol (see Table 1) was to ...

Table 1: Exercise progression; suppl. was initiated in w7 after lactate test

"evaluate effects of the use of supplementation with leucine or a mixture of BCAAs in trained rats submitted to an exercise-induced protocol of glycogen depletion.

Furthermore, we attempted to investigate muscle and liver biochemical parameters that were not performed in the previous study in order to elucidate the role of BCAAs in glycogen depletion. " (Campos-Ferraz. 2013)

In other words: The researchers wanted to find out whether or not leucine would exert identical, less or more pronounced effects on muscle glycogen use and endurance performance in rodents that the full spectrum of branch-chained amino acids, i.e. leucine, valine and isoleucine.

Contrary to what bro-science and the shiny ads of the supplement industry are suggesting, the scientists' fundamental hypothesis was that the BCAAs supplementation would impair the rodents endurance capacity, because the branched-chain amino acids would be used in muscle to yield acetyl-CoA. This, in turn could reduce the activity of the glucose-alanine cycle, by which the muscles are supplied with alanine-derived glucose from the liver and (once the BCAAs got burne) result in an earlier onset of fatigue.

BCAAs are "glycogen depleters"?!

If you take a look at the data Campos Ferraz et al. gathered in the testing sessions at the end of the supplementation period, in the course of which the rats received an oral gavage of 166mg/kg per day (in human terms this would be ca. 3-3.5g per day) of BCAAs or leucine, it is quite obvious that the  the leucine group had a significantly lower muscle and liver glycogen degradation ratios than the BCAA group.

Figure 1: Liver & mucle glycogen degradation and time to exhaustion (expressed relative to placebo); muscle TCA intermediate content and enzyme activity / concentration (Campos-Ferraz. 2013)

Compared to the placebo group, only the ratios were different.  While the placebo group had the lowest liver glycogen use and a high muscle glycogen use, the supplemental leucine induced a shifted from muscle to liver glycogen and did thus exert muscle specific glycogen sparing effects.

As the researchers point out, these observations stand in line with their original hypothesis: Leucine can spare a significant amount of muscle and liver glycogen and thus produce a highly significant increase in resistance to exhaustion compared to the mixture of BCAAs (P<0.001).

This is not the first study to cast a bad light on BCAA supplementation. As a SuppVersity veteran, you will remember my November 2012 article "Chronic High Dose BCAA Supplementation Reduces Endurance Performance by 43%" | read more, as well as the more recent investigation into the  "Neurotransmitter Depleting Effects of Branched Chain Amino Acids (BCAAs) and Their Potential Ergolytic, Anxiogenic & Depressive Downstream Effects" | read more.

If we compare the endurance performance of the leucine rodents to that of the placebo group, this does yet cast a slight shadow on the overall image of the glorious ergogenic, and, even more so, the purported performance enhancing effects of BCAAs. Despite measurable differences in the time to exhaustion, the actual endurance increase in response to the leucine supplement is relatively small.
 
If you take another look at the data in Figure 1 you will probably notice the significant increase in TCA cycle intermediates (citrate and malate) in the BCAA group. These changes provide further evidence that the provision of all three branch-chain amino acid emphasized the use of glucose as a main substrate to sustain the endurance activity.

"Mouse vs. man": Can we ignore the differences in BCAA metabolism?


At this point, it may however be about time to point out that the activity of the BCAA catabolizing enzyme branched-chain keto acids dehydrogenase complex (BCKD) in humans is quite different from that in rats.

"In the latter [the rat], liver BCKD is almost completely unphosphorylated (activated) in basal state, making it possible to metabolize more rapidly BCKA from the portal blood; in humans, BCKD in liver is normally phosphorylated (inactivated) in order to spare BCAAs for protein synthesis." (Campos-Ferraz. 2013)

In other words: While rodents use BCAAs mostly as an energy source, the human body spares them as a potential protein anabolic.

In view of the fact that the BCAAs are not used to the same degree as an alternative substrate in the human vs. the rodent liver, it is actually not very surprising that the results of the study at hand appear to conflict with data from a previous study by the same laboratory (Gualano. 2011). In the corresponding experiment, Gualano et al observed measurable increases in exercise capacity and lipid oxidation in human subjects during endurance exercise after muscle glycogen depletion in response to the provision of 300mg/kg BCAAs per day.

So, the study is totally irrelevant, right? Not really, no. The fact that we are not able to use BCAAs as a readily available energy source like rodents does after all not mean that they must necessarily have the opposite effects on us. In fact, you all know that the vast majority of studies investigating the beneficial effects of BCAAs on endurance performance in humans yielded a null-result (!) - despite the fact fact that generations of researchers have been convinced that the inhibition of tryptophan uptake must blunt the exercise induced onset of fatigue (learn more in the articles cited in the red box).

Don't forget the endurance reducing increase in glucose usage that appears to be caused by isoleucine (and maybe valine) can also be beneficial: "The Glucose Repartioning Effects of Isoleucine" | read more.

The actual new information this study brings to the table is thus not that BCAAs are not ergogenic. It's rather the previously overlooked leucine induced acceleration of the glucose alanine cycle in liver. It is the activation of this (catabolic!) powerhouse by the means of which leucine "might have an interesting use in physical performance in prolonged or submaximal exercise, where muscle glycogen stores are more likely to be depleted" (Campos-Ferraz. 2013). It should be noted, though, that these effects are probably only observed after the glycogen levels are fully depleted - after an intense workout, towards the end of a race or after an fasted training - in those situations, the performance benefits may even be more more significant than in the study at hand.

Reference:

• Campos-Ferraz PL, Bozza T, Nicastro H, Lancha AH Jr. Distinct effects of leucine or a mixture of the branched-chain amino acids (leucine, isoleucine, and valine) supplementation on resistance to fatigue, and muscle and liver-glycogen degradation, in trained rats. Nutrition. 2013 Nov-Dec;29(11-12):1388-94.
• Gualano AB, Bozza T, Lopes De Campos P, Roschel H, Dos Santos Costa A, Luiz Marquezi M, et al. Branched-chain amino acids supplementation enhances exercise capacity and lipid oxidation during endurance exercise after muscle glycogen depletion. J Sports Med Phys Fitness 2011;51:82–8