Glycine for Your Gains? Glycine Boosts Protein Synthesis (80%), Reduces Protein Degradation (-30%) in Muscle Cells

If glycine worked in athletes as it did in pigs or even isolated muscle cells, in which scientists recently observed a dose-dependent increase in protein synthesis (up to 80%) and reductions in protein degradation (-30%), it would be a go-to supplement for dieting athletes.

Glycine is not exactly the most popular amino acid supplement in the health and fitness community. With recent studies by a group of Chinese and American scientists from the State Key Laboratory of Animal Nutrition in China and the Texas A&M University, this may change - rightly so?

Only recently Wang et al. were able to show that dietary glycine supplementation enhances skeletal muscle growth in young pigs (Wang. 2014a,b) - an excellent model of human metabolism. In view of the fact that the mechanism has hitherto not been fully understood, it was difficult to judge whether the smallest possible amino, i.e. glycine, could / would be useful for trainees as well.

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With their latest study, the same team of scientists, this time with KaiJi Sun as their lead author (Sun. 2016), provide an experimentally supported explanation of the anabolic effects of glycine - an explanation that is not just convincing but would also suggest that glycine could be an interesting supplement for bodybuilders and fitness freaks, as well as their grandparents.

Table 1: Physiological functions of glycine in animals and humans (Wang. 2013).

How's that? Well, while we are dealing with a cell line study, the scientists' analysis I'd like to remind all of you who are about to click on "comment" and complain that this is a follow-up study on the muscle-specific effects of an amino acid of which we have known for quite long that it is important for animal and human health.

Where's the human study in trained athletes? Early evidence for its ergogenic effects comes from Buchman, et al. (1999) who actually wanted to use glycine as a control for the beneficial effects of arginine on marathon runners, and then realized that it was glycine that enhanced the subjects' performance. Similar, albeit inconsistent effects have been reported for GPLC, i.e. Glycine Propionyl-L-Carnitine which improved resistance training performance in Jacobs, et al. 2009, but failed to yield ergogenic effects in a long-term follow-up with negative effects on aerobic acid with high dose (4.5g/day) supplementation of GPLC. Another glycine-compound with mixed evidence of its ergogenic prowess is GAKIC, i.e. glycine-arginine--ketoisocaproate, which has been shown to improve the performance of repeated cycling sprints in Buford, et al. (2004) - a result that could not be confirmed in a follow-up by Beis, et al. (2011), though.

Collagen hydrolysate, a high glycine protein (right bars), turned cata- into anabolism in a recent study in older women on low protein diets whey (left bars) did not (Hays. 2009).

At this point, the existing evidence is thus clearly insufficient to recommend glycine itself or any of the supplement industry's favorite glycine compounds as go-to supplements for athletes. The only thing that comes close is a recent study showing a decrease in nitrogen excretion (a marker of, you guessed it, protein breakdown) and thus reversal of the negative nitrogen balance with high glycine collagen protein - and by the way, the negative nitrogen balance persisted when the older women who participated in this 2-week study consumed a whey supplement (see figure on the left | Hays. 2009).

It is also an amino acid of which the previously cited studies showed that its use as a dietary supplement will (Wang. 2014b)...

• increased small-intestinal villus height (could reverse leaky gut and will improve nutrient transport), intestinal transport of glycine, plasma concentrations of glycine and GSH, as well as whole-body growth and protein accretion, while 
• reducing plasma concentrations of ammonia, urea, and glutamine, 

in a dose-dependent manner. Evidence the scientists rightly interpreted as follows: "Based on these lines of compelling evidence, we conclude that glycine is a nutritionally essential amino acid for maximal growth and development of milk-fed young pigs" (Wang. 2014b).

Figure 1: Effects of different doses of glycine on muscle anabolism (blue, left axes) and catabolism (gray, right axes) in C2C12 myotubes - all differences were highly significant (p = 0.001 | Sun. 2016).

With their latest study on C2C12 muscle cells, the scientists were now able to explain both the increased whole-body growth and protein accretion and the reduced ammonia, urea and glutamine concentrations, of which at least the former are clear signs of protein catabolism and thus potential muscle loss - actual gains, however, have yet to be observed outside of model organisms like pigs - also to quantify how much glycine it will take to achieve similar effects as they were observed in vivo.

MAFbx/atrogin-1 and MuRF1 gene activity mediates the degradation of protein and have an inhibitory effect on protein synthesis (Foletta. 2011).

Do I need glycine if I consume whey or BCAAs? With its ability to do both, promote mTOR and inhibit the MuRF1 and atrogin-1 gene expression and thus protein loss in C2C12 myoblasts, Glycine appears to have everything many people ascribe to BCAAs.

The one important difference, however, is that BCAAs don't affect the catabolic muscle genes and catabolism (learn more). While they appear to ameliorate the exercise-induced increase MuRF-1 total protein (Borgenvik. 2012), their effect on the gene expression of the atrophy regulators (see Figure on the right) in muscle cells is nowhere to be found.

Similarly, providing more protein in form of whey protein may further augment your protein synthesis via mTOR increases. What it does not do, however, is to affect the MuRF1 and atrogin-1 mRNA levels. Accordingly, it will only target one side of the net protein accretion equation (gains = existing muscle + muscle synthesis - muscle loss) - or as Stefanetti who studies this effect with different types of resistance exercise in 2014 write: "While WPH supplementation with ECC and CONC training further increased muscle hypertrophy, it did not have an additional effect on mRNA or protein levels of the targets [MAFbx/atrogin-1 and MuRF1] measured" (Stefanetti. 2014).

As discussed in the previous infobox, though, the one, decisive study that would proof practically relevant anti-catabolic effects glycine in healthy human beings, not isolated muscle cells, is not available yet. If you want to target muscle catabolism with supplements, carnitine (Keller. 2013) and HMB appear to be better, but likewise not fully proven candidates | Comment!

References:

• Borgenvik, Marcus, William Apró, and Eva Blomstrand. "Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle." American Journal of Physiology-Endocrinology and Metabolism 302.5 (2012): E510-E521.
• Buchman, A. L., et al. "The effect of arginine or glycine supplementation on gastrointestinal function, muscle injury, serum amino acid concentrations and performance during a marathon run." International journal of sports medicine 20.05 (1999): 315-321.
• Buford, BRITNI N., and Alexander J. Koch. "Glycine-arginine--ketoisocaproic acid improves performance of repeated cycling sprints." Med Sci Sports Exerc 36 (2004): 583-587.
• Hays, Nicholas P., et al. "Effects of whey and fortified collagen hydrolysate protein supplements on nitrogen balance and body composition in older women." Journal of the American dietetic association 109.6 (2009): 1082-1087.
• Jacobs, Patrick L., et al. "Glycine propionyl-L-carnitine produces enhanced anaerobic work capacity with reduced lactate accumulation in resistance trained males." Journal of the International Society of Sports Nutrition 6.1 (2009): 1.
• Keller, Janine, et al. "Supplementation of carnitine leads to an activation of the IGF-1/PI3K/Akt signalling pathway and down regulates the E3 ligase MuRF1 in skeletal muscle of rats." Nutrition & metabolism 10.1 (2013): 1.
• Mascher, Henrik, et al. "Effects of essential amino acid supplementation on markers for anabolic and catabolic response to resistance exercise in female subjects." The FASEB Journal 24.1 Supplement (2010): lb278-lb278.
• Stefanetti, Renae J., et al. "Influence of divergent exercise contraction mode and whey protein supplementation on atrogin-1, MuRF1, and FOXO1/3A in human skeletal muscle." Journal of Applied Physiology 116.11 (2014): 1491-1502.
• Sun, KaiJi, et al. "Glycine Regulates Protein Turnover by Activating Akt/mTOR and by Inhibiting MuRF1 and Atrogin-1 Gene Expression in C2C12 Myoblasts." The Journal of Nutrition (2016): jn231266.
• Wang, Weiwei, et al. "Glycine metabolism in animals and humans: implications for nutrition and health." Amino acids 45.3 (2013): 463-477.
• Wang, Weiwei, et al. "Glycine stimulates protein synthesis and inhibits oxidative stress in pig small intestinal epithelial cells." The Journal of nutrition 144.10 (2014a): 1540-1548.
• Wang, Weiwei, et al. "Glycine is a nutritionally essential amino acid for maximal growth of milk-fed young pigs." Amino Acids 46.8 (2014b): 2037-2045.

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

References:

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

True or False? Glycine & Proline Supplements Ramp Up Collagen Synthesis & Improve Joint Health. Plus: The Tripeptide Advantage of Collagen Hydrolysates

The "Paleo" cult has repopularized eating and preparing your own (Chicken) bone broth, but will this also help with bone and cartilage health?

Although you're probably thinking of collagen as the stuff that's important for joint health, its implications in human health are more far-reaching than most of us believe.

In fact, collagens are the most abundant group of organic macro-molecules in human and animal body. Because of their tensile strength, they perform numerous structural functions within the body - specifically in connective tissues which include among other tissue also organs as your heart, your intestines, your lungs and the parenchymal organs like the liver and the kidneys and even the fibrous matrix of skin and blood vessels.

As I already said, collagens are yet by far best known as structural components of the protein matrix of the skeleton and its related structures, like bones, teeth, tendons, cartilage and ligament, which bring us back to the original question that bothered me after assuring Chris who emailed me asking about the necessity of taking glycine and proline supplements in the absence of any other protein (my answer was "that's bullocks"): Do glycine and problem supplements even help with collagen synthesis and joint health? Or is the supplement vendor next door the only person who benefits?

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We do have evidence (from rodent studies) that the ingestion of low molecular weight (=small peptides) collagen hydrolysates with intact glycyl-prolyl-hydroxyproline tripeptides that actually make it through the gut into the bloodstream and will increasee the organic substance content and decreased the water content of the left femur (Watanabe-Kamiyama. 2009). Previous studies had already shown hat the content of an orally administered gelatin hydrolysate will be incorporated into the cartilage tissue of rats (Oesser. 1999). Similar observations have been made by Iwai et al. for human volunteers and porcine gelatine hydrolysate, as well.

"After the oral ingestion, the peptide form of Hyp significantly increased and reached a maximum level (20-60 nmol/mL of plasma) after 1-2 h and then decreased to half of the maximum level at 4 h after the ingestion. Major constituents of food-derived collagen peptides in human serum and plasma were identified as Pro-Hyp. In addition, small but significant amounts of Ala-Hyp, Ala-Hyp-Gly, ProHyp-Gly, Leu-Hyp, Ile-Hyp, and Phe-Hyp were contained." (Iawai. 2005)

If we assume a similar physiological effect as it was observed by Watanabe-Kamiyama in rodents, the ingestion of (large) quantities of gelatine could thus very well, after it's hydrolysation in the gut, have similar effects on human cartilage tissue as the collagen hydrolysate that was used in the Watanabe-Kamiyama study.

Table 1: Summary of Structure and Recovery of Food-Derived Collagen Peptide in Human Serum or Plasma after Oral Ingestion of Gelatin Hydrolysates (Iawai. 2005).

With respect to the occurence of glycyl-polyl-hydroxproline tripeptides, of which the Watanabe-Kamiyama study suggests that they may be responsible for the beneficial effects on cartilage synthesis it should yet be said that it occurred in human plasma only after the ingestion of chicken, but not in porcine collagen in the Iawai study (see Table 1). If that's no coincidence, HARIBO, which is usually made with porcine gelatine is no "collagen builder", a real chicken soup, cooked with bone, on the other hand, could be.

Given that your stomach is working properly a nice paleo bone broth (preferably from chicken bone) could thus produce similar results as a collagen hydrolysate of which a recent review in Current Medical Research and Opinion says that its ingestion stimulates a statistically significant increase in synthesis of extracellular matrix macromolecules by chondrocytes.

There is more to collagen hydrolysates than joint health: In 2009 Saito et al. were able to show that fish collagen hydrolysates affect lipid absorption and metabolism in rats and may be useful in suppressing the transient increase of plasma triglycerides (Saito. 2009). Moreover, Spanish researchers showed that the daily dietary intake of hydrolyzed collagen seems to have a potential role in enhancing bone remodeling at key stages of growth and development in 60 children (9.42±1.31 years) who had been randomly assigned to either placebo or collagen (+ calcium) supplementation. In spite of these benefits, the ingestion of corresponding supplements is not necessary for people with healthy collagen metabolism who exercise regularly and eat clean.

Figure 1: Physician rated (top) and subject-rated (bottom) improvement in joint pain walking (left) and standing (right) in the Clark study (Clark. 2008).

The authors, researchers from the University of Illinois College of Medicine at Chicago and the University of Kiel in Germany add:

"These findings suggest mechanisms that might help patients affected by joint disorders such as OA. Four open-label and three double-blind studies were identified and reviewed; although many of these studies did not provide key information – such as the statistical significance of the findings – they showed collagen hydrolysate to be safe and to provide improvement in some measures of pain and function in some men and women with OA or other arthritic conditions." (Bello. 2006)

Subsequent studies such as Benito-Ruiz et al. (2009) or Clark et al. who evaluated data from 97 athletes from a varsity team or a club sport in Pennsylvania support Bello's conclusion (see Figure 1).

Similar beneficial effects were also observed by  et al. in a more recent study with "normal" subjects with articular pain in response to 1,200mg/day of collagen hydrolysate (Bruyère. 2012). When we're looking into the effects of single amino acids, however, things look different. If they're ingested separately, glycine and proline are not going to form a tripeptide in the course of the digestive process. And while they may still serve as a raw material for the endogenous synthesis of such peptides the chance that they actively promote the synthesis of new collagen is slim.

Biologically active tripeptides, not just glycine & proline is what you want!

Bottom line: Collagen hydrolysates with intact tripeptides seem to have a beneficial effect on collagen synthesis. Classic broth and gelatine, both best made from chicken bones (absorption data on beef is not available), could have beneficial effects on collagen synthesis. In view of the chance that and rate at which the physiologically relevant  glycyl-prolyl-hydroxyproline tripeptides (see image to the right) are produced during the natural digestion process it does yet appear certain that you would have to garble down tons of it on a daily basis to actually trigger collagen synthesis and not just to do what individual amino acids could probably do as well: provide the necessary substrates without actually accelerating collagen synthesis.

Chris' original question whether you'd have to take glycine and proline supplement on their own and in the absence of any other proteins and amino acids would thus actually be obsolete (you shouldn't take them at all), but I guess it may be worth mentioning that doing that, i.e. taking them on their own will only increase the "risk" of both being used by the liver as a substrate for glyconeogenesis (proline for example has the 3rd highest potential for gluconeogenesis 75% of the most glycogenic amino acid, i.e alanine; cf. Ross. 1967) - especially if you top "taking them on their own" with "taking them during a fast".

References:

• Bello, Alfonso E., and Steffen Oesser. "Collagen hydrolysate for the treatment of osteoarthritis and other joint disorders: a review of the literature." Current Medical Research and Opinion® 22.11 (2006): 2221-2232.
• Benito-Ruiz, P., et al. "A randomized controlled trial on the efficacy and safety of a food ingredient, collagen hydrolysate, for improving joint comfort." International journal of food sciences and nutrition 60.S2 (2009): 99-113. 
• Bruyère, Olivier, et al. "Effect of collagen hydrolysate in articular pain: a 6-month randomized, double-blind, placebo controlled study." Complementary therapies in medicine 20.3 (2012): 124-130.
• Iwai, Koji, et al. "Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates." Journal of agricultural and food chemistry 53.16 (2005): 6531-6536.
• Oesser, Steffen, et al. "Oral administration of 14C labeled gelatin hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57/BL)." The Journal of nutrition 129.10 (1999): 1891-1895. 
• Ross, B. D., R. Hems, and H. A. Krebs. "The rate of gluconeogenesis from various precursors in the perfused rat liver." Biochem. J 102 (1967): 942-951.
• Saito, Masataka, et al. "Effect of collagen hydrolysates from salmon and trout skins on the lipid profile in rats." Journal of agricultural and food chemistry 57.21 (2009): 10477-10482.
• Watanabe-Kamiyama, Mari, et al. "Absorption and effectiveness of orally administered low molecular weight collagen hydrolysate in rats." Journal of agricultural and food chemistry 58.2 (2009): 835-841.

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 ;-)

Arginine + Glycine a Synergistic Duo for Gut Health

Lately, "gut health" has become a topic of interest way beyond colonics and irritable bowel syndrome. Scientists begin to understand more and more how important an intact intestinal system is for nutrient absorption, infection defense and general health. The results of a very recent study published in the Journal of Colerectal Disease (Picano. 2011) are thus relevant for all of us.

In an experiment with rats Picano et al. found that a combination therapy of l-arginine and glycine inhibited irradiation induced damage to the colon walls of the animals:

Stereologic analysis showed that irradiation induced a reduction of the total volume of the colon wall of group II and III animals compared to healthy controls, but not of group IV animals supplemented with glycine. The mucosal layer of the irradiated animals of all groups was reduced compared to healthy group I animals, but supplementation with L-arginine and glycine was effective in maintaining the epithelial surface of the mucosal layer.

Although I hope none of you is treated (or otherwise exposed) to similar doses of ionizing radiation, it certainly won't hurt your gut health to have an eye on your dietary intake of these two amino acids, in order to maintain optimal gut health.