Wednesday, November 7, 2012

The Dipeptide Advantage!? +43% Muscle Glycogen With Whey Hydrolysate Compared to Matched Amino Acid Mix

The bigger the choice, the harder it is to choose. If you had to pick just one, go for the concentrate, if you want to afford two, get an isolate or hydrolysate and a casein protein.
"Whey is still the way to go." I believe I wrote, or at least thought just that only a couple of days ago, when I wrote about the wheat gluten hydrolysate in the last installment of the SuppVersity Science Round Up Seconds (click here to read all previous installments). Unfortunately, there is not just one way... ah, pardon me, I do of course mean "whey", there are many! And in order to completely confuse their customers supplement companies will spike their concentrates, isolates and even hydrolysates with free form amino acids, or - which is even more confusing - advertise their BCAA and EAA products as being made from whey protein. I mean, who cares if the damn molecules are unbound and indistinguishable, anyway? But let's get to the point, a soon to be published study from Japan could yield at least some insights into demonstrable and purported benefits of one over the other.

Whey hydrolysate or simply its aminos, what gets pro-glycogen job done?

Based on results from previous experiments in the course of which the researchers had found that the acute provision carbohydrate + whey protein hydrolysate (WPH) had superior effects on muscle glycogen repletion compared to iso-nitrous amounts of BCAAs (Morifuji. 2010a) Kanda et al. speculated that chronic supplementation with whey protein hydrolysate (WPH) should elicit similarly beneficial effects and could help them clarify the underlying mechanism of this glycogen boosting effects of WPH. To this ends, the researchers put a group of mice on diets that differed only terms of the protein composition of the diet.
  • Table 1: Composition of the test diets
    The control group's exclusive protein source was the casein from the standard chow.
  • The whey amino acid (WAA) group received a chow, where 50g of the casein protein was replaced by an iso-nitrogenous amount free amino acids that was matched to the amino acid composition of the whey hydrosolate.
  • The whey hydrolysate group received 20% of the original casein from the standard chow in form of a whey hydrolysate from Meiji Co., Ltd.
In the course of the 4-week study period, body weight and food intake of the animals were measured on a weekly basis. The mRNA expression, protein levels, and phosphorylation of glycoregulatory enzymes were measured in the gastrocnemius muscle. All rodents performed a regular 30-min swimming exercise protocol  in a fancy adjustable-current water pool five times per week and had to 'survive' a weekly (endurance-)performance test in the course of which they had to swim to absolute failure (defined as being drowning for 7s, already).
Figure 1: Food intake, liver weight, and visceral fat (left), as well as time to total fatigue during swimming exercise (right) of the rodents on control, WAA and WPH diets  (data adapted from Kanda. 2012)
Contrary to the data from this test, the body composition markers in figure 1 do not show any significant inter-group differences (positive or negative) the same goes for the food intake and the liver weight, as well as for the total body weight of the mice, which was totally identical (not shown in figure 1). The said swimming performance of the WAA and WPH group, on the other hand, were 32% and 48% higher than in the control group.

More glycogen synthetase = more glycogen content = more endurance

Now, despite the fact that this increase did - for whatever reason - not reach statistical significance, Kanda et al. are convinced that this increase in endurance must be a direct consequence of the increase in glycogen storage, which has been observed by Evans and Hughes in 1985 (Evans. 1985), already, and has been confirmed numerous times thereafter.
Figure 2: Glycogen content and glycogen synthetase levels (GS), as well as mRNA expression of glycogen synthase I and the ratio of phosphorylated to unphosphorylated GS (data based on Kanda. 2012)
And, as you can see, the actual data in figure 2 clearly confirms this hypothesis. The mice who had received whey protein hydrolysate (WPH) in their diets for the whole 4-week study period had significantly (p < 0.05) higher muscle glycogen levels than their peers in the control group (73%) and still more than 40% more total glycogen than the mice who had been fed the amino acid enriched chow (WAA). Quite impressive, right? And all that is just a consequence of a peptide induced elevation in glycogen synthetase and it's activity, which is indicated by the lower ratio of phosphorylated (=incative) to un-phosphorylated (=active) levels of this tightly regulated enzyme.

There is more to whey than BCAAs

The total amount or activity of the glucose transporter (GLUT-4) as well as the hexokinase activity (which figures in the phosphorylation of sugars) were not different between treatments and though the dreaded gluconeogenesis in the liver was not measured it is, given the high amount of carbohydrates in the diets of the rodents, very unlikely that the higher susceptibility of "fast" protein sources to be oxidized, when no other nutrients are available, played a significant role in the 'pro-glycogenic' of whey hydrolysate (after all the rodents consumed the protein as part of their chow), so that the most likely explanation for the superiority of the whey protein hydrolysate over the iso-nitrous amino acid mixtures remains their peptide content. In this regards, the authors of the study remark:
"The amino acid compositions of the two diets used in this study contained equal amounts of BCAA and leucine; however, muscle glycogen accumulation varied between diets. This result strongly suggests that not only the BCAA content but also the molecular form of BCAA found in the protein source might be important for muscle glycogen storage." (Kanda. 2012)
Kanda et al. do then refer to a previous study, in which his group had been able to demonstrate that BCAA-containing peptides in WPH, which have been shown to be markedly elevated (meaning they are not digested) after the consumption of whey protein hydrolysates (Morifuji. 2010) in a follow up study on human beings, do actually have the ability to stimulate the rate of glucose uptake in vitro (Morifuji. 2009).

Figure 3: Insulin response after the ingestion of 12.5g of either soy or whey protein or their respective hydrolysates (Morifuji. 2010)
"And what about insulin?"

I know that this question is now on your minds and in way you are right the only slight caveat you have to keep in mind before you blindly follow the scientists' outspoken advice to consume "carbohydrates mixed with WPH [to] enhance sport performance by increasing glycogen storage" (Kanda. 2012) would in fact be be the increased insulin response. Of the latter, the scientists found in the aforementioned 2010 human trial (Morifuji. 2010) that it is ~70% more pronounced in the first hour after the the ingestion of 12.5mg of whey protein hydrolysate and, once more compared to regular whey protein, still ~17% higher over the whole 2h period (the different proteins you see in figure 3 were all ingested on an empty stomach after an overnight fast by the 10 normal-weight subjets; the data I mentioned refers to the AUC values on the bottom of figure 3).

The insulin 'spike' is not necessarily a problem. At the right time in the right person it can even be highly beneficial.

Nevertheless, the results of the study at hand to actually confirm that before you invest in all sorts of useless pills, it may make more sense to make sure that you a) have more than just a single protein powder in your supplement arsenal and that you b) have been reading enough SuppVersity articles to be able to use them properly ;-) ... What? You still don't know how? Well assuming you have no problems with high insulin levels and are mainly interested in building muscle, you could start out with 20-30g whey + 15-25g casein (depending on your body size and needs) after a workout (see "Whey & Casein Work Hand in Hand for Muscle Protein Anabolism") and a 40g casein shake pre-bed (see "3.2kg of Lean Mass Overnight").

By the whey *lol*, I suppose you will see similar benefits from a whey isolate, although this would have to be tested. What does not need any tests, on the other hand is that you better make sure you don't forget the carbs! I mean, what is your body supposed to use as a substrate for the increased glycogen synthetase activity, if you are depriving yourself of carbohydrates? The protein your liver converts to blood glucose? Yeah, what a glorious idea... whatare your brain and your other organs going to use then? Ketones? No way, if you are pounding tons of fast acting glucogenic amino acids in form of protein shakes.

  • Evans, W. J.; Hughes, V. A. Dietary carbohydrates and endurance exercise. Am. J. Clin. Nutr.1985, 41 (5, Supplement), 1146−1154.
  • Kanda A, Morifuji M, Fukasawa T, Koga J, Kanegae M, Kawanaka K, Higuchi M. Dietary Whey Protein Hydrolysates Increase Skeletal Muscle Glycogen Levels via Activation of Glycogen Synthase in Mice. J Agric Food Chem. 2012 Oct 31.
  • 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. 2009, 55(1), 81−86.
  • Morifuji, M.; Kanda, A.; Koga, J.; Kawanaka, K.; Higuchi, M. Post-exercise carbohydrate plus whey protein hydrolysates supple-mentation increases skeletal muscle glycogen level in rats.Amino Acids 2010a, 38(4), 1109−1115.
  • 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. 2010b, 58(15), 8788−8797.