|How much of the protein you can eat and how much of it you need two keep the status quo are very different questions.|
What we are going to deal with today is protein breakdown, or the purported general anticatabolic effect of high protein diets. To this ends, we will be taking a closer look at the ubiquitin proteasome system (UPS) response to constant energy deficits (ED) at varied dietary protein intakes before and after the consumption of a high protein meal replacement. An issue, by the way, that was also addressed in a paper that has been published a couple of days ago in the peer-reviewed scientific journal FASEB (Carbone. 2013).
|No, this is not a deja vue! You've actually read about the same experiment, yet a different portion of the results back in June, in one of my previous articles on optimal protein intake / supplementation | read more|
- 0.8g/kg body weight of protein (RDA)
- 1.6g/kg body weight (2 -RDA), or
- 2.4g/kg body weight (3 -RDA)
and to be physically fit [peak oxygen intake (Vo2peak) 40–60 ml/(kg ·min)].
The subjects followed the prescribed dietary protocol for a whole month (31 days), went into a tightly controlled 10-day weight maintenance phase and started fasting for 21 days, immediately thereafter (Note: To keep the protein intake stable, the dietitians who planned and prepared the meals for all study participants had to prepare meals with a significantly higher relative protein content),
In this study: 30% energy restriction +10% physical activity = "fasting"
Just to avoid any confusions: The above, i.e. a 30% reduction in energy intake and a 10% increase in physical activity, is what John W. Carbone and his colleagues talk about, when they use the word "fasting". It does not mean that the subjects have been sitting in one of those tents, where you can actually measure the energy expenditure for 21-days eating nothing, but their finger and toe nails.
An important note on the accuracy of calculated: While Carbone et al. state that their -30% intake, +10% expenditure protocol will produce an energy deficit of 40%, I'd hope that you as seasoned SuppVersity students see through the futility of calculations like these and let go off figures that signify a degree of exactness that's simply not there.In view of the fact that the term "fast" is contemporarily used to designate "diets" that are really low in energy (up to ZERO calories, intermittently), I suspect I should also mention that I personally would call the last 21 days of the study the "diet" not "fasting" phase.I mean, 30% reduced energy intake and +10% physical activity? We all know that the health and physique 90% of our fellow men and women would benefit from this regimen.
Muscle biopsies and enzyme expressions
The scientists took muscle biopsies before (fasted) and 2h after (fed) the ingestion of "a commercial nutrition supplement (Boost; Nestlé HealthCare Nutrition, Florham Park, NJ, USA)" with a total energy content of 480 kcal and 20 g of protein to assess the degree of intracellular proteolysis on day 10, i.e. right before the "fast", and day 31, i.e. right after the "fast", of which Carbone et al. write that it was based on individualized menus that were administered under supervision, to ensure compliance.
"To maintain fitness, the volunteers performed resistance type physical activity 3 d/wk, and daily endurance-type exercise, at levels comparable to those they had reported in their prestudy activity logs. To minimize the potential that an unaccustomed training stimulus would influence skeletal muscle outcomes and to ensure accuracy, research personnel closely controlled and monitored the intensity and volume of physical activity. For resistance-type exercise, the volunteers performed 1 single-joint movement per major muscle group (3 sets of 15 repetitions), using workloads determined during the prestudy period. The intensity of the endurance-type activity (40 – 60% VO2peak) was based on prestudy measurements, and verified by indirect calorimetry (ParvoMedics) and the corresponding heart rate during familiarization trials conducted before the study, and by the heart rate reserve-method throughout the interventio." (Carbone. 2013)The 10% increase in physical activity was achieved by an increase in the length of the daily endurance training sessions that would accommodate for the corresponding increase in energy expenditure
|Figure 1: Changes in lean body mass and fat mass (kg) that occured during the 21-days on 40% energy restricted diets with varying amounts of dietary protein in it (based on Pasiakos. 2013)|
What's the more important "-bolism": Cata- or ana-bolism?
In contrast to their previous paper that focused exclusively on the increase in muscle protein synthesis, this "follow up"* discards the influx of dietary protein into the muscle of the subjects and focuses on the proteolytic enzymatic response to the diet (*I assume both were filed at the same time, but the one in FASEB was published ahead of print, while the one at hand did not).
|Figure 2: Enzymatic activities for 26S 1(A), 26S 2(B), 26S 5(C), and caspase-3 (D). Open bars, weight maintenance; solid bars, energy restriction.|
I mean, you would expect that the activity of the catabolic enzymes would vary depending on the protein content of the diets, wouldn't you? No? Well, maybe you did expect that the response to the 20g protein shake the subjects consumed would have an effect on their expression (black bars in Figure 2)?
You didn't? Tthat's awesome, you must be a genius, 'cause nothing of that actually happened. In other words, the expression of proteolytic enzymes did not in any way or form depend on the total amount of protein, the healthy young subjects in the study at hand consumed on a daily basis - once, twice or thrice the RDA of 0.8g/kg body weight.
Take home message #1 -- Your daily total protein intake has no effect on the expression of catabolic enzymes in your musculature. Even your total energy intake has little effect on the expression of catabolic enzymes in your musculature.And if we are honest, most of us would probably also have expected a much more pronounced difference in these markers of muscle catabolism, when comparing the weight maintenance to the fasting phase (white vs. black bars) - a difference that existed for some yet not all of the ubiquinase enzymes, but was statistically significant for none of them.
|Figure 3: mRNA expression of the Ub ligases MuRF1, atrogin-1 and TNF-alpha (Carbone. 2013)|
"But how come that both are increased with higher protein intakes?"
Actually you know the answer already. It's after all not just take home message #2 of today's SuppVersity article, but has been addressed in many previous articles on total protein intake and the effects of protein supplementation here at the SuppVersity, as well.
Take home message #2 -- The more protein you eat the more wasteful your body will be (note: this does not mean that the net protein retention does not increase, but it means that you will see diminishing at intakes of thrice the RDA even non-existent returns; cf. "Are you Protein Wheysting?")So what's left do discuss then? Ah, right, aside from the proteolytic enzymes, the scientists also tested for changes in the expression of TNF-alpha & co and observed that the expression of TNF- mRNA and activation of NF- B1 increased as protein intake exceeded the RDA.
This increase in TNF-alpha and NF-B1 may at first surprise you - TNF [tumor necrosis factor] and consequent NF- B activation are, after all, generally associated with increased muscle proteolysis. If you look back at "take home message #2", however, you'll realize that this, i.e. an increase in muscle proteolysis is exactly what's going on, in the 2x and 3x RDA groups. It is thus also logical that this increase in tumor necrosis factor occurs only in the fed state - a state, when dietary protein is abundantly available.
- Carbone, J. W., Margolis, L. M., McClung, J. P., Cao, J. J., Murphy, N. E., Sauter, E. R., ... & Pasiakos, S. M. (2013). Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis. The FASEB Journal, 27(12), 5104-5111.
- Gomes, M. D., Lecker, S. H., Jagoe, R. T., Navon, A., & Goldberg, A. L. (2001). Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proceedings of the National Academy of Sciences, 98(25), 14440-14445.
- Pasiakos SM, Cao JJ, Margolis LM, Sauter ER, Whigham LD, McClung JP, Rood JC, Carbone JW, Combs GF Jr, Young AJ. Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial. FASEB J. 2013 Jun 5. [Epub ahead of print].
- Schakman, O., Dehoux, M., Bouchuari, S., Delaere, S., Lause, P., Decroly, N., ... & Thissen, J. P. (2012). Role of IGF-I and the TNFα/NF-κB pathway in the induction of muscle atrogenes by acute inflammation. American Journal of Physiology-Endocrinology And Metabolism, 303(6), E729-E739.
- Witt, S. H., Granzier, H., Witt, C. C., & Labeit, S. (2005). MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination. Journal of molecular biology, 350(4), 713-722.