No Advantage of Bolus Ingestion of EAAs in Young Men!? Cereal Bread Not Better for Weight Control. Saturated Fat & the Heart. Plus: Serine for Your Weekend Alcohol Binge!

The "muscle full effect" indicates you don't have to consume 4 scoops at once.

With the publication of the latest issue of The Journal of Nutrition came a handful of interesting scientific papers I will briefly introduce in today's SuppVersity Nutrition Science Update.

The corresponding studies deal with the link of saturated fat to heart disease (Puaschitz. 2014), the effects of proteinogenic amino acid serine (one of the non-essential amino acids) on homocysteine metabolism in a rodent model of alcoholic fatty liver disease (Sim. 2014).

And when we're through with those, we will take a closer look at the effects of cereal enriched breads on the appetite ratings and postprandial glucose, insulin, and gastrointestinal hormone responses related to hunger and satiety in healthy men and women (Gonzalez-Anton. 2014), and the "muscle full effect", or rather limits to maximal protein synthesis in man (Mitchell. 2014).

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

• Saturated fat and your heart - Right from the Haukeland University Hospital in Norway comes a new study that investigated the associations between self-reported dietary SFA intake and risk of subsequent coronary events and mortality in patients with coronary artery disease (CAD).
  
  The study included patients who participated in the Western Norway B-Vitamin Intervention Trial and completed a 169-item semiquantitative food-frequency questionnaire after coronary angiography - 2412 patients, total, 81% men, 19% women with a mean age of 61.7 y.
  After a median follow-up of 4.8 y, a total of 292 (12%) patients experienced at least one major coronary event during follow-up.  And while a gigh intake of SFAs was associated with a number of risk factors at baseline, "there were no significant associations between SFA intake and risk of coronary events [age- and sex-adjusted HR (95% CI) was 0.85 (0.61, 1.18) for the upper vs. lower SFA quartile] or any secondary endpoint. Estimates were not appreciably changed after multivariate adjustments" (Puaschitz. 2014).
  

  

  Figure 1: Hazard ratios according to % saturated fat intake of total energy intake compared to minimal saturated fat intake (HR = 100%) in 2412 subjects (Puaschitz. 2014).

  In other words, if you ask researchers from Northern Europe, their answer to the question, whether our high intake of saturated fats is the reason we are dying prematurely from heart disease is "no". This stand in line with a recent review of the current evidence by O'Keffee et al. who point highlight that the different results (which often depend on the country, where the studies are conducted) may be attributable to the fact that "not all SFA are created equal and the food sources of SFA". Accordingly the researchers from the King's College in London, the Luke’s/Roosevelt Hospital, the New York Nutrition Obesity Research Centre and the Columbia University in New York recommend that "individual characteristics of the SFA, such as chain length, should be considered in dietary recommendations" (O’Keeffe. 2014)... and I would like to add: In every future study, as well.
  
  I mean, this and the foods from which the subjects in the study at hand got the majority of their saturated fat intake may well be the reason that there was a statistically significant correlation between high fat intakes and the occurernce of coronary artery disease (remember: all participants had CAD, already) in the cohort Western Norway B-Vitamin Intervention Trial.
• L-Serine as super-supplement for binge drinkers? At least in rodents the provision of 200mg/kg body weight (for humans this would be ~1.2-1.5g/day) serine in the diet led to an attenuation of alcohol-induced increases in serum homocysteine and hepatic triglyceride (TG) concentrations (>5-fold in the control mice) by 60.0% and 47.5%, respectively.
  

  

  Figure 2: Liver triglyceride levels, serum ALT and serum homocysteine levels in control mice (C) and "binge drinking mice" (EV) with and without 20mg/kg (ES20) and 200mg/kg (ES200) serine in their diets (Sim. 2014)

  Moreover, in the chronic ethanol study, l-serine also decreased hepatic neutral lipid accumulation by 63.3% compared with the ethanol group and ramped up the glutathione and S-adenosylmethionine content of the liver by 94.0% and 30.6%, respectively.
  
  If we assume that serine is only half as powerful, when it is given to humans, I would recommend you drink your Vodka Red Bull with serine in the future ;-)
• Super-satiating cereal enriched breads - I guess "super-satuating" is an exaggeration, but there is no doubt that the addition of variety of cereal flours (wheat, oat, and spelt) and 22% dried fruits (figs, apricots, raisins, and prunes) to regular bread lead to a significant improvement of appetite control by reducing hunger and enhancing satiety in 30 healthy adults (17 men and 13 women) aged 19–32 y with body mass index of 19.2–28.5 who participated in an experiment that was conducted at the University of Granada in Spain (Gonzalez-Anton. 2014).
  

  

  Figure 3: The hormonal changes would indicate increased satiety, the subjects reported increased satiety, but their 4h energy intake was identical in both condition (Gonzalez-Anton. 2014)

  Whether the decrease in prospective consumption and increased satiety is enough to have long-term benefits on weight control is yet questionable, because the subsequent ad libitum energy intake in a 4 h period after the ingestion of the "enriched" bread did not differ from that in the control condition, even though the postprandial blood glucose, insulin, ghrelin, were lower and the pancreatic polypeptide AUC (an indicator of satiety) was higher than with the control bread.
  
  Speaking of insulin: In view of the fact that the latter actually is a satiety hormone and its release is closely related to glucagon-like peptide (GLP) 1 and gastric inhibitory polypeptide (GIP) where the AUC (areas under the curve) were lowered as well, it's eventually maybe not too surprising that the "enriched" bread was not better than the regular one.
• Muscle full? What's limiting protein Synthesis? Scientists from the Clinical, Metabolic, and Molecular Physiology, MRC–Arthritis Research UK Centre of Excellence for Musculoskeletal Ageing Research at the University of Nottingham and the Royal Derby Hospital in the United Kingdom recently determined the effect of Bolus (=all the aminos at once) vs. Spread EAA feeding in young men, hypothesizing that muscle-full is regulated by a dose-, not delivery profile–, dependent mechanism; and what they found was surprising for us - not for the researchers, though:

  

  Figure 4: Even though the study was conducted in young men, the overall dosage of 15g may potentially have had an effect on the outcome. On the other hand: If you "overdose" it would actually be more likely for spread protein ingestion to have superior effects. Against that background the "low" dose of "only" 15g of pure EAAs is not an argument that would falsify the results of the study at hand (Mitchell. 2014)

  "Despite distinct plasma and muscle profiles, Bolus feeding provided no anabolic advantage over Spread feeding (or vice versa); these findings are in keeping with our hypothesis of there being an intrinsic muscle-full state in young men at rest.
  
  Bolus feeding led to rapid aminoacidemia with a brisk upstroke and high peak plasma EAA and leucine concentrations. Spread feeding, by comparison, resulted in lower, later peak concentrations. Despite this, identical MPS responses were observed, even with the same latency (of ~90 min) and amplitude.
  
  Furthermore, with both feeding strategies, basal MPS was observed 180 min after consumption of either Bolus or the initial Spread doses. This preceded the peak Spread plasma EAAs, in keeping with the onset of a muscle-full state.

  As the scientists point out, their results do thus "suggest that, in healthy young men, it is dose dependent mechanisms that regulate the size of the anabolic response to feeding and that this response" and that this dose-dependent anabolic response "is not perturbed by later arriving, lower-amplitude aminoacidemia." The researchers also highlight hat it would seem "vital to have such a mechanism in place"; because of the "stability of muscle mass from year to year in healthy younger populations" (Mitchell. 2014). Eventually, the differences may well be explained by the existence of three distinct phases in the postprandial period, the scientists argue:
  

  

  Figure 5: Absolute changes in FSR from fasted (2120 to 0 min) to fed (0 to 240 min) (A), actual FSRs (B) and plasma EAA and insulin concentrations, phospho- 4EBP1 Thr65/70 and muscle protein synthetic rates, normalized to their own data spans shown on the same axis (C and D) in young men after consumption of 15 g of mixed-EAA meals by Bolus or Spread treatment. The black arrows represent ingestion of 15 g EAAs once, and the gray arrows represent ingestion of 3.75 g EAAs 4 times (Mitchell. 2014)

  "After the onset of essential aminoacidemia, a latent period exists when a significant negative arteriovenous EAA balance is detectable (Mitchell. 2013) but incorporation of EAAs into newly synthesized myofibrillar proteins is not. The existence of a similar latent period in response to Bolus and Spread EAA ingestion suggests that providing time for adequate intracellular EAA accumulation, even with rapid aminoacidemia with Bolus, is crucial before MPS can be ‘‘switched on.’’ After this latent period, a transient stimulation in MPS, lasting ;90 min (Bohé. 2001), occurs before the onset of the muscle-full state restores basal MPS despite sustained, near-peak postprandial EAA availability" (Mitchell. 2014).

  Put simply, it takes long enough for the muscle protein synthesis to gain full speed to incorporate all the amino acids the healthy subjects received in 4x45min boluses.
  
  Practically speaking this does not necessarily mean that you should give up your previous protein feeding strategies. With intact proteins, of which you know that they are more than the sum of their EAA parts (see "Whey Beyond Brawn"), studies by Moore et al. (2012 | learn more) and Burke et al. (2012 | learn more) yielded different results... albeit with less frequent biopsies that were taken across the postprandial period and thus a lower temporal resolution that does not exclude that said studies simply overlooked the dose-dependency of the muscle-full effect Mitchell et al. demonstrate in the study at hand.

10+ Things You Probably Didn't Know Whey Protein | more

So what are the take home messages from today's research update? I guess the one you will be most interested in, is the related to the Mitchell study which indicates that protein timing and / or the importance of bolus ingestions may previously have been overrated - at least in the short run. We should not forget, after all, that this is a result that would stand in line with Alan Aragon's & Brad Schoenfeld's recent review (Aragon. 2014  on nutrient timing which found a significant effect for the amount of protein people consume, but no evidence of the purported importance of protein timing.

This is yet not the only myth that is tumbling. The idea of heart disease triggering saturated fats and the notion that you could make bread a superfood by adding cereals and dried fruits did not get away unscathed either. With the impressive effects of serine in the rodent study by Sim et al. (2014), we do have another myth to bother with - one of which I would like to remind you that it has to remain a myth until the results have been confirmed in human beings | Comment on Facebook!

References:

• Aragon, Alan Albert, and Brad Jon Schoenfeld. "Nutrient timing revisited: is there a post-exercise anabolic window." J Int Soc Sports Nutr 10.1 (2013): 5.
• Bohé, Julien, et al. "Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids." The Journal of physiology 532.2 (2001): 575-579.
• Burke LM, Hawley JA, Ross ML, Moore DR, Phillips SM, Slater GR, Stellingwerff T, Tipton KD, Garnham AP, Coffey VG. Preexercise aminoacidemia and muscle protein synthesis after resistance exercise. Med Sci Sports Exerc. 2012 Oct;44(10):1968-77.
• O’Keeffe, Majella, and Marie-Pierre St-Onge. "Saturated Fat and Cardiovascular Disease: A Review of Current Evidence." Current Cardiovascular Risk Reports 7.2 (2013): 154-162. 
• Mitchell, William Kyle, et al. "Development of a new Sonovue™ contrast‐enhanced ultrasound approach reveals temporal and age‐related features of muscle microvascular responses to feeding." Physiological reports 1.5 (2013). 
• Mitchell, William Kyle et al. "A Dose- rather than Delivery Profile–Dependent Mechanism Regulates the ‘‘Muscle-Full’’ Effect in Response to Oral Essential Amino Acid Intake in Young Men."J. Nutr. February 1, 2015
• Moore DR, Areta J, Coffey VG, Stellingwerff T, Phillips SM, Burke LM, Cléroux M, Godin JP, Hawley JA. Daytime pattern of post-exercise protein intake affects whole-body protein turnover in resistance-trained males. Nutr Metab (Lond). 2012 Oct 16;9(1):91.
• Puaschitz et al. "Dietary Intake of Saturated Fat Is Not Associated with Risk of Coronary Events or Mortality in Patients with Established Coronary Artery Disease." J. Nutr. February 1, 2015 jn.114.203505
• Sim, et al. "l-Serine Supplementation Attenuates Alcoholic Fatty Liver by Enhancing Homocysteine Metabolism in Mice and Rats." J. Nutr. February 1, 2015 jn.114.199711.

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 ;-)