Monday, February 11, 2013

Don't Judge a Protein by Its Amino Acid Content: 17% Higher Protein Synthesis With Whey vs. Free Form Amino Acids

Take a look at the FRS of sedentary rats in figure 2, compare them to those who endured the 2h swimming torture and tell me "rest" was not important to build muscle (read more about resting and recovery).
Those of you who have read about the recent revelation that the hitherto often overlooked branched-chain amino acid isoleucine is in fact the major driving force behind the nutrient repartitioning effects of BCAAs (it shuttles the glucose into the muscle instead of the adipose tissue, read more), will probably remember that isoleucine was not the only molecule in that study with the ability to increase GLUT-4 expression and skeletal muscle glucose uptake. Its 2-amino-acid peptide cousins Lue-Ile and Ile-Leu, both combinations of leucine and isoleucine, exerted similar effects. Against that background the latest results, Atushi Kanda and his co-workers from the Meiji Company Ltd. in Kanagawa, as well as Kentaro Kawanaka an Mitsuri Higuchi from the Niigata University of Health and Welfare and the Waseda University present in the British Journal of Nutrition probably won't come as a total surprise to most of you.

Compared to EAAs alone, whey induces a 17%  higher protein synthesis

To determine whether there would be a significant difference in the appearance of amino acids (essential or not) in the blood stream of the Sprague Dawley rats, the researchers used in the study, and to elicit whether this would translate into an identical protein synthetic response the scientists fed their semi-fasted rodents after 2h of swimming in a water-filled barrel a test-solution containing either
Figure 1: Nutrient composition (Kanda. 2013).
  • whey protein hydrosylate (WPH)
  • free form amino acids (WAA)
  • carbohydrates (CHO)
That this is not exactly the most "anabolic" workout is probably nothing I would have to tell you, right? I still want to mention it, because it reduces the significance of the results, or rather the size of the differences that were observed in the study at hand.
Why does the choice of the exercise protocol matter? What we are actually most interested in is obviously the protein synthetic response after a strength training workout, right? A brief intense hypertrophy workout would however provide a nutrient independent stimulus for protein synthesis. For 2 hours of exhaustive swimming this is not the case, so that any protein supplement will initially have to set the protein synthetic machinery into gear. Now, we got to do to do some math to understand why this makes a difference: Let's quantify our effects for whey vs. AA as 117 and 100 arbitrary units, respectively. If we add those to a baseline level of 0 a.u. (= protein synthesis after 2h of swimming) we get a 17% difference between totals of 117 and 100 a.u. If we add the same 117 and 100 a.u. to a baseline level of say 50 a.u. (hypothetical value after a brief resistance training workout) we will observe total levels of 167 and 150 and thus an increase of only 11% - got it?
The iso-energetic "test-meals" the scientists had prepared covered 15% of the daily energy requirements of the laboratory rats and the whey and amino acid supplement had completely identical amino acid make-ups (see figure 2) with the only difference being that the AA supplement contained all of them in their pure crystalline forms, while the whey protein hydrosylate contained mostly small peptides (bonds of amino acids), with an average chain length of 3.64.
Figure 2: Fractional protein synthesis (left), amonio acid glucose and insulin concentrations in the blood and expression of mTOR, 4E-BP1 and S6K1 in skeletal muscle in response to CHO, whey protein hydrosylate (WPH) or identical free form amino acids (AA; Kanda. 2013)
As the data in figure 2 shows, the allegedly "minor" difference did produce a significantly more pronounced fractional protein synthesis rate of roughly 0.7%/day vs. only 6%/day (+17%) in those rodents who had been receiving the whey + CHO solution after their involuntary 2-h "workout" - and that despite the fact that the amino acid levels in the blood of both groups were virtually identical!

Identical amino acid levels in the blood, but profoundly different fractional protein synthesis

The underlying reasons for this difference can be found in the differential response of the protein signaling cascade to free form amino acids and the small peptides in the whey protein hydrolysate, where the phosphorylation of mTOR (the notorious mammalian target of rapamycin and the central and locally acting trigger of skeletal muscle protein synthesis) and its downstream targets, the eukaryotic initiation factor 4E-binding protein 1 (4EBP-1) and ribosomal protein S6 kinase (S6K1) levels, was significantly enhanced compared to both the carbohydrate only and the essential amino acid groups (figure 2, right).
Don't forget that you cannot expect the same mTOR response as on day one, after weeks of intense training. Take some time off and reset your protein anabolic response (learn more about detraining and the difference to tapering)
Post-exercise ingestion of glucose plus WPH caused a significant increase in phosphorylated mTOR levels compared with AA or CHO. The WAA group also showed a significant increase in phosphorylated mTOR compared with the CHO group. The WPH group had greater phosphorylation of 4E-BP1 compared with both the AA and CHO groups. The WAA group also exhibited a significant increase in phosphorylated 4E-BP1 compared with the CHO group. Phosphorylation of S6K1 at Thr389 and Thr421/Ser424 was greater in the WPH group than in the AA and CHO groups. The AA group also demonstrated greater phosphorylation of S6K1 at both these sites compared with the CHO group.
I guess, I could hardly have said it more concise or could I? Well, I guess I could. "WPH > WAA > CHO wrt to FSR", or in a complete sentence: The maximal fractional protein synthesis was achieved with the whey protein hydrosylate, the isolated amino acids came a close second, and the' carbohydrate only' supplement did not have any effects on protein synthesis, at all.

"So what is it that makes whey special"

If you like it gross, you should consider taking some Deer Blood Isolate. Crazy idea? Well maybe, but when I just wanted to find a study with the exact peptide composition of WPH I came up with a 2013 paper by scientists from the Wildlife Institute of the Heilongihang Province in (you guessed it) China. Who state that deer blood hydrosylates "could be a potent food antioxidant" in their recently published paer in the Journal of Advanced Materials Research (Shuai Wang. 2013). Certainly an alternative to deer antler velvet, right *rofl*?
Actually I have answered the above question in the introduction already. Rather than just being subject to further breakdown, the short peptides that arise in the process of hydrolysation, are taken up by their own transporter system in the gut and will, just like the phrase "build muscle!" deliver a different message than the words "build" and "muscle" alone to muscle cells; or, as the scientists say it: "The WPH used in the present study contained active components that are superior to leucine itself for increasing muscle protein synthesis" (Kanda. 2013).

Just as I did in the introductory paragraph with the study by Morato et al., Kanda et al. refer to previous study by their research group (Morifuji. 2009), in which they
" [...] demonstrated that BCAA-containing peptides in WPH, such as Ile-Val, Leu-Val, Val-Leu, Ile-Ile, Ile-Leu, Leu-Ile and Leu-Leu, increased the rate of glucose uptake in isolated rat skeletal muscle"  (Kanda. 2013)
Their rationale is that it would only logical that the same BCAA-containing peptides that promote muscle glycogen synthesis may also "act as bioactive peptides in both glycogen and muscle protein synthesis in skeletal muscle" (Kanda. 2012) and that this would be relevant for both man and rodent has been demonstrated in a previous study from the same group already (Morifuji . 2010).

 Ex-Mr. Universe, Manohar Aich, turns 100 and swears by the synergy of whey & casein in milk (read more)
Are isolated or specifically engineered BCAA containing peptides the future? In view of the fact that the total amount of dipeptides that actually make it into the blood stream is low and reaches maximal concentrations that are 13-40x lower than those of leucine and other free form amino acids (based on unpublished data by the same group), the profound differences in protein synthesis, the scientists observed do certainly suggest that isolated or even better specifically engineered branched-chain amino acid containing small peptides are going to be the future of protein supplementation.

Until the future turns into the present, however, regular whey proteins and - as reluctant as I am to admit that - particularly the partially hydrolyzed whey protein hydrolysates will remain the #1 choice for post workout supplementation. That a combination of whey + casein and a subsequent whole foods meal is probably still going to yield the largest net protein accretion is a different matter and one I would finally like to be addressed in a single study investigating the full time-frame to the next workout, so that we would no longer have to rely on hypotheses based on various oftentimes hardly comparable isolated experiments.

  • Kanda A, Nakayama K, Fukasawa T, Koga J, Kanegae M, Kawanaka K, Higuchi M. Post-exercise whey protein hydrolysate supplementation induces a greater increase in muscle protein synthesis than its constituent amino acid content. Br J Nutr. 2013 Feb 7:1-7. [Epub ahead of print] 
  • Morifuji M, Koga J, Kawanaka K, Higuchi M. Branched-chain amino acid-containing dipeptides, identified from whey protein hydrolysates, stimulate glucose uptake rate in L6 myotubes and isolated skeletal muscles. J Nutr Sci Vitaminol (Tokyo). 2009 Feb;55(1):81-6.
  • Morifuji M, Ishizaka M, Baba S, Fukuda K, Matsumoto H, Koga J, Kanegae M, Higuchi M. Comparison of different sources and degrees of hydrolysis of dietary protein: effect on plasma amino acids, dipeptides, and insulin responses in human subjects. J Agric Food Chem. 2010 Aug 11;58(15):8788-97. 
  • Zeng J, Zhu H, Kong J. Antioxidant Properties of Deer Blood Hydrolysate and the Possible Mode of Action. Advanced Materials Research. 2013; 1435:634-638.