Leucine s Effect on Protein Muscle Synthesis through the Mechanistic Target of Rapamycin (mtor) By Brandon Lukas Lee Dr. William Proulx, PhD, RD, CDN NUTR 340-01 Due Date: 12/15/15
TABLE OF CONTENTS Introduction... 3 Review of Literature... 3 Leucine a determinant of postprandial skeletal muscle protein synthesis in adult rats... 3 Physical activity and leucine in muscle protein synthesis in partially pancreatectomised rats... 4 Supplementation of leucine: effect on myofibrillar protein synthesis at rest and post- resistance exercise in men... 5 Leucine ingestion after resistance exercise prolongs myofibrillar protein synthesis in older men... 7 Postprandial muscle protein synthesis is higher after leucine-enriched supplement compared to a diary-like product in healthy older people... 7 Conclusion... 9 References... 9 2
INTRODUCTION Skeletal muscle, the heart, kidneys, diaphragm, adipose tissue and other organs are responsible for all three branch-chain amino acids through branch-chain amino transferases, located in the mitochondria and cytosol. One branch-chain is of particular interest, leucine. Leucine is a vital amino acid in muscle protein synthesis and has a direct correlation to mechanistic target of rapamycin (mtor) (4). In that correlating relationship, when there is leucine withdrawal, muscle protein synthesis is brought to a halt; whereas a diet rich in leucine promotes positive anabolic effects. Studies conducted seek to further confirm the fact that the outcome of leucine on mtor-mediated signaling in the cells of muscles can be accomplished by a simple single concentration of leucine (4). REVIEW OF LITERATURE I. Leucine and Skeletal Muscle Protein Synthesis Norton et al. hypothesized that the dietary effect of leucine would be greatest during situations when muscle protein synthesis is deregulated and the protein content of the meal is in short supply (1). The first experiment investigated post-meal in muscle protein synthesis, plasma leucine, and adaptation factors in forty-two rats that were fed meals from different sources of protein: whey protein (n= 11), wheat (n= 10), egg (n=11) and soy (n=10). Experiment one s duration was fifteen days and involved killing the rats on the last day for a close examination of blood and tissue samples. Furthermore, in experiment two, the rats had their wheat gluten meals supplemented with leucine in order to figure out if equaling the amount of leucine in whey and wheat meals would result in similar peak rates of postprandial muscle protein synthesis. Experiment two (as in experiment one) was a single meal study and involved euthanizing the rats. Experiment two involved five to six rats (based on group). Experiment one resulted in an 3
increase in plasma leucine escalation in whey or egg groups although in the wheat and/ or soy groups, no such increase was discovered. In experiment two, the magnitude of the post-prandial change was largely diverse with the highest concentration observed at 30 minutes and then decline at 90 and 135 minutes. This showed that a short supply of protein within the situation of a small meal; stimulation of muscle protein synthesis is reliant on intake of leucine to activate the mammalian target of rapamycin (mtor). II. Physical activity and Muscle Protein Synthesis In 2013 Wilkinson et al. reviewed the effect of leucine on human skeletal muscle protein metabolism (3). The focus of the research was to study the effects of HMB (β-hydroxy-βmethylbutyrate) on the proteins within mammalian muscle turnover through mtor and to compare it with leucine. Outcomes show that small amounts of two to three grams of leucine or its metabolite HMB react with a rise in muscle protein construction by inhibiting the mtor complex, further reinforcing the benefits of leucine. A study by Seino et al. used partially pancreatectomised rats in order to imitate hyperinsulinemia/ hyperglycemia to mimic a child with inadequate insulin administration (2). The hypothesis of this study was to establish two things. The first was to observe if physical activity would raise the synthesis of the proteins in muscle and muscular growth in young rats. The second was to see if voluntary daily physical activity would alter the mtor1 signaling feedback of skeletal muscle to intense leucine feeding. The rats were divided into one group out a selection of three, mock or false operated (n= 18), sedentary pancreatectomised (n=14) and exercise pancreatectomised. The rats were given leucine gavage and modified flooding dose techniques to measure muscle protein synthesis. Moreover, data showed elevation in 4
phosphorylated and total eukaryotic initiation factor (eif), 4E binding protein 1 (4E-BP1), serine/threonine protein kinase Akt and ribosomal protein S6 kinase 1 (S6K1). These measurements were completed in regards to Akt and mtor1. The results show the active rats having increased muscle volume and fibre areas the same to that of false groups. Moreover, the sedentary rats showed a 20-30% loss in muscle fibre locations. Muscle protein build responding to leucine gavage was defective in sedentary rats (by about 65%) but not in active rats compared to the sham group. Markers of mtorc1 were increased in shams (by two- and nine fold, correspondingly) and in active rats (1.5- and fourfold, correspondingly) although did not have the same results in sedentary rats. In conclusion, with regular physical activity, there can be improvements in mtorc1 signaling response and an increased rate of compartmental protein synthesis in muscle in feedback to leucine supplementation. III. Leucine on Muscle Protein Synthesis at Rest and After Exercise Churchward-Venne et al. conducted research in 2012 based on adding leucine to a diet low in protein and its effect on muscle protein construction at rest and after resistance training in males (6). The hypothesis of the study was that leucine would prove to be the muscle protein synthesis equivalent of whey protein in two different experimental groups. The two experimental groups were the feeding group and the combined feeding and resistance exercise group. Alternately there was a second hypothesis, that leucine would have an outcome of a rise in muscle protein synthesis in the feeding group and the resistance combined feeding group; however, the response would be less than whey protein and leucine combined because of the lower leucine content. Furthermore, changes in the phosphorylation condition of protein targets of the Akt-mTOR pathway and in the mrna bounty of certain amino acid carriers were 5
investigated. In the study twenty-four recreationally active, young adult male volunteers were used (6). Subjects underwent one-sided strength testing of the extensor muscles of the knee approximately one to two weeks after the experimental infusion trial. Diet design was based on the Harris-Benedict equation and were accommodated for modest activity levels (1.4-1.6) based on individual s subject physical activity history. To summarize, the three nutrient treatments were whey protein (with 25g of whey and 3 g of leucine), leucine (6.25g of whey isolate with 3g total of free form leucine) and EAA- leucine (6.25g whey protein isolate added with to EAA s but without leucine). The results showed that there was no change among treatment groups for the subject s strength testing (6). In addition, the blood samples taken from the subjects resulted in leucine showing a considerable but fleeting increase following leucine as compared to whey protein with the whey showing a more conservative but sustained a positive rise nonetheless. Moreover, the results showed p-mtor being substantially elevated three hours postprandial in the feeding group, and one, three and five hours later in the resistance and feeding combination. In conclusion, supplementation of suboptimal dosage of whey protein with leucine or a combination of EAAs without leucine is an active method to encourage rates of muscle protein synthesis after a meal. Additionally, leucine in small amounts is all, that is needed in young ablebodied people abundant supply of other EAAs are added. Churchward-Venne conducted a similar research to the previously mentioned on based on leucine supplementation in low protein drinks for young men (5). The study added leucine to low protein drinks for the subjects for both at rest and after exercise to observe the effects on muscle protein synthesis. Subjects completed unilateral knee-extensor resistance and then the participants were spilt into five different categories based on how much leucine was added to their whey protein drink. The most effective category proved to be 6.25 g of whey protein with 3 6
g of total leucine added (the highest amount of leucine provided to any one group). In addition, postprandial periods resulted in a constant rise in muscle protein build. This study also reinforces leucine s effect on the muscle protein synthesis. IV. Leucine-Enriched and Exercise on Protein Synthesis and Amino Acid Transport Dickinson et al. looked at the effect of post fitness training leucine supplementation and myofibrillar protein synthesis, but in older men (7). In this study sarcopenia and resistance exercise were studied along with sarcopenia prevention. The hypothesis of the study was to see if leucine-enriched essential amino acids ingestion after resistance exercise would enhance myofibrillar protein synthesis in fifteen able-bodied elderly men (aged 72 years ± 2 y). The participants were considered as individuals with recreational activity in their lifestyles. Participants were assigned to one of two groups; the control and the leucine groups. Participants did various types of resistance exercises and were given essential amino acids (10g) and leucine (experimental 1.85g, control 3.5g); blood samples were later taken. The results showed that post exercise (one hour) ingestion of 10g of essential amino acids containing either 1.85 or 3.5 g of the leucine supplement had analogous effects on myofibrillar protein synthesis soon after (twofive hours) resistance exercise (7). Furthermore, the experiment showed that post-exercise ingestion of increased assembly of leucine extended the increase duration in myofibrillar protein synthesis; this continued up to twenty-four hours after the resistance exercise. In short the outcome of leucine on myofibrillar protein synthesis is effective on mtor within older men. V. Whey protein and Postprandial Muscle Protein Synthesis 7
Luiking et al. experimented on muscle protein syntheis comparing the effects of a leuicne-enriched supplemnt and a dairy by-product in able-bodied elderly adults (8). The aim of the study was focused on whether the whey protein supplmental nutrition drink with enriched in leuicne would be active than a usual diary product to prompt muscle protein synthesis in older able-bodied adults. In addition, the study predicted that exercise would continue to add to postpradial muscle protein build. Methods for the study involved nineteen older adults, sixty years and older with a BMI between 21-30kg.mˉ² (8). The research was designed as a randomized, controlled, double blind study where the volunteers received a single bolus of a high whey protein, leucine-enriched supplement or an iso-caloirc milk protein control after unilateral resistance training. The experimental group (whey, leuicne supplement) consisted of 20g whey protein, 3g total leucine, 150 kcal with nine subjects. Moreover, the control group (milk protein) consisted of 6g milk protein with ten subjects. A tacer infusion protocol with L-[ring-C6]- phenylalanine and regular blood and muscle sampling was used to measure postprandial mixed msucle protein factional rate over the course of four hours. The results presented data resprenting a higher postprandial muscle protein synthesis rate in the experiemental group (0.0780 ± 0.0070 %/h) as compared with the control group (0.0574 ± 0.0066%/h) based on a typical single serving of a conventional dairy product. The ending of the study concludes that overall in healthy older adults that after resistance exercise a nutritional supplement with added leucine can better promote muscle protein sysnthesis. Another study that observed the effect of added leucine in older adults was conducted by Casperson et al. (9). In this study the focus was on the prevention of sarcopenia. Over the course of two weeks the subjects had meals supplemented with leucine and had metabolic studies conducted on them on day one and day fifteen. The study used infusion of L-[ring-C6]- 8
phenylalanine and additionally measured markers of protein synthesis (e.g. mtor), body composition, mixed muscle fractional synthesis rate pre and post a low protein/ carbohydrate assumed mealtime. The study concluded that leucine supplementation in older adults can yield positive results in musclar protein sysnthesis in reply to low protein feeding. Conclusion Muscle protein build is the adaptive reaction to diet and exercise and is a continuous progress. Skeletal muscle mass is decided on a biological balance of protein synthesis and protein degradation. In the progress of muscle protein build exists the mechanistic target of rapamycin (mtor) and is the key component in the control of protein synthesis and protein degradation (10). mtor happens as two protein complexes, mtor complex 1 containing raptor (mtor1) and mtor complex 2 containing rictor (mtor2). The essential amino acid leucine has been seen to inhibit the mtor complex to promote muscle growth/ synthesis and prevent muscle wasting in relation to certain diseases and sarcopenia (9). Based on past research, recent discoveries and general scientific skepticism, it is widely understood that the study of leucine and its role in the natural biological process still needs more research. References: 1) Norton, L. E., Wilson, G. J., Layman, D. K., Moulton, C. J., & Garlick, P. J. (2012). Leucine content of dietary proteins is a determinant of postprandial skeletal muscle protein synthesis in adult rats. Nutrition & Metabolism, 9(1), 67-75 9p. doi:10.1186/1743-7075-9-67 2) Serino, A. S., Adegoke, O. A., Zargar, S., Gordon, C. S., Szigiato, A. A., Hawke, T. J., & Riddell, M. C. (2011). Voluntary physical activity and leucine correct impairments in muscle protein synthesis in partially pancreatectomised rats. Diabetologia, 54(12), 3111-3120. doi:10.1007/s00125-011-2296-0 3) Wilkinson, D. J., Hossain, T., Hill, D. S., Phillips, B. E., Crossland, H., Williams, J., &... Atherton, P. J. (2013). Effects of leucine and its metabolite β-hydroxy-β-methylbutyrate 9
on human skeletal muscle protein metabolism. The Journal Of Physiology, 591(Pt 11), 2911-2923. doi:10.1113/jphysiol.2013.253203 4) Areta, J. L., Hawley, J. A., Ye, J., Chan, M. S., & Coffey, V. G. (2014). Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells. Nutrition Research, 341000-1007. doi:10.1016/j.nutres.2014.09.011 5) Churchward-Venne, T. A., Breen, L., Di Donato, D. M., Hector, A. J., Mitchell, C. J., Moore, D. R., &... Phillips, S. M. (2014). Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: a double-blind, randomized trial. American Journal Of Clinical Nutrition, 99(2), 276-286 11p. doi:10.3945/ajcn.113.068775 6) Churchward-Venne, T. A., Burd, N. A., Mitchell, C. J., West, D. D., Philp, A., Marcotte, G. R., &... Phillips, S. M. (2012). Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men. The Journal Of Physiology, 590(Pt 11), 2751-2765. doi:10.1113/jphysiol.2012.228833 7) Dickinson, J. M., Gundermann, D. M., Walker, D. K., Reidy, P. T., Borack, M. S., Drummond, M. J., &... Rasmussen, B. B. (2014). Leucine-Enriched Amino Acid Ingestion after Resistance Exercise Prolongs Myofibrillar Protein Synthesis and Amino Acid Transporter Expression in Older Men. Journal Of Nutrition, 144(11), 1694-1702. doi:10.3945/jn.114.198671 8) Luiking, Y. C., Deutz, N. E., Memelink, R. G., Verlaan, S., & Wolfe, R. R. (2014). Postprandial muscle protein synthesis is higher after a high whey protein, leucineenriched supplement than after a dairy-like product in healthy older people: a randomized controlled trial. Nutrition Journal, 9) Casperson, S. L., Sheffield-Moore, M., Hewlings, S. J., & Paddon-Jones, D. (2012). Original article: Leucine supplementation chronically improves muscle protein synthesis in older adults consuming the RDA for protein. Clinical Nutrition, 31512-519. doi:10.1016/j.clnu.2012.01.005 10) Ham, D. J., Caldow, M. K., Lynch, G. S., & Koopman, R. (2014). Review: Leucine as a treatment for muscle wasting: A critical review. Clinical Nutrition, 33937-945. doi:10.1016/j.clnu.2014.09.016 10