METHODS FOR ENHANCING MUSCLE PROTEIN SYNTHESIS DURING ENERGY DEFICIT

BACKGROUND

[0001] The present disclosure relates generally to health and nutrition. More specifically, the present disclosure relates to methods for enhancing muscle protein synthesis during energy deficit.

[0002] Negative energy balance or energy deficit can be achieved through reduced energy intake and/or increase energy expenditure and leads to fat mass (FM) loss. A reduction in FM is a common goal for both athletic performance and improved health outcomes (Hansen D. Dendale P, Berger J, van Loon LJ, Meeusen R, The effects of exercise training on fat-mass loss in obese patients during energy intake restriction. Sports Med 2007; 37: 31-46: Mettler S, Mitchell N, Tipton KD, Increased protein intake reduces lean body mass loss during weight loss in athletes. Medicine and science in sports and exercise 2007; 42: 326-337). However, when a reduction in FM is achieved by energy restriction alone, it results in the concomitant loss of fat free mass (FFM), predominantly skeletal muscle (Parr EB, Coffey VG, Hawley JA, 'Sarcobesity' : a metabolic conundrum. Maturitas 2013;74: 109-113). Given that the quality and quantity of skeletal muscle is a major determinant of athletic performance, whole body metabolic rate and functional capacity throughout the life span, nutritional and exercise strategies to prevent or minimize loss of FFM while losing fat mass are crucial.

[0003] If potential decrease in basal (resting) rates of muscle protein synthesis are not accompanied by a concomitant reduction in muscle protein breakdown, then energy deficit could lead to an imbalance in resting protein turnover that could favor the net loss of skeletal muscle proteins. Indeed, prolonged energy deficit-induced body weight loss can result in up to 60% fat free mass (Paskiakos 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 Journal 2013). In contrast, exercise has been shown to attenuate the loss of lean body mass that can occur with periods of energy deficit (Stiegler P, Cunliffe A, The role of diet and exercise for the maintenance of fat-free mass and resting metabolic rate during weight loss. Sports Medicine 2006;36: 239-262).

[0004] Ingestion of protein/amino acids increases skeletal muscle protein synthesis, an effect that is enhanced by prior resistance exercise (Moore DR, Robinson MJ, Fry JL, Tang JE, Glover EI, Wilkinson SB, Prior T, Tarnopolsky MA, Phillips SM, Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. American Journal of Clinical Nutrition 2009;89: 161-168). Whether skeletal muscle exhibits 'anabolic resistance' to exercise and protein ingestion following short-term energy deficit has not been determined. Nor has the effect of increased protein availability through post-exercise ingestion.

[0005] Currently it is unknown if the anabolic effects of resistance exercise are decreased during periods of energy deficit. Therefore, there is a need to further understand the effects of energy deficit on the regulation of muscle protein synthesis. There is also a need to show the benefit of exercise and post-exercise protein intake on muscle protein synthesis during periods of energy deficit. More specifically, there is a need to show the impact on skeletal muscle translation initiation signaling, mRNA expression and rates of muscle protein synthesis during energy deficit.

SUMMARY

[0006] The present disclosure is related to methods for enhancing muscle protein synthesis. Specifically, the present disclosure is directed to recovery of the attenuation in muscle protein synthesis observed with periods of acute dietary energy restriction with ingestion of whey protein (in different amounts) following resistance exercise. As discussed above, there is no existing evidence that the established beneficial effects of protein ingestion on maintaining/enhancing muscle protein synthesis following resistance exercise are equally effective during periods of energy deficit. In contrast, Applicants have developed methods that provide a beneficial outcome for individuals in short-term ( e.g., 5 days) energy deficit for maintenance of the anabolic response to protein ingestion following exercise. Therefore, the present disclosure provides a unique outcome that is highly applicable to sports nutrition consumers that intentionally undergo periods of energy deficit to optimize body composition during which they aim to avoid any skeletal muscle loss.

[0007] More specifically, the present disclosure provides methods of off-setting the muscle catabolic state observed during energy deficit by provision of high quality protein (e.g., 15-30 g) post-exercise. The present disclosure also provides a method of augmenting the anabolic effects of resistance exercise with protein ingestion during short- term energy deficit in a dosage that is higher than those reported for individuals in energy balance (e.g., 20-25g) ((Moore DR, Robinson MJ, Fry JL, Tang JE, Glover EI, Wilkinson SB, Prior T, Tarnopolsky MA, Phillips SM, Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. American Journal of Clinical Nutrition 2009;89: 161-168).

[0008] Further, the present disclosure provides for restoration/augmentation of muscle protein synthesis, which is an early measure of muscle growth and adaptation (Cermak NM, Res PT, de Groot LC, Saris WH, van Loon LJ. Protein supplementation augments the adaptive response of skeletal muscle to resistance-type exercise training: a meta-analysis. American Journal of Clinical Nutrition (2012)). Indeed, no evidence currently exists to show the beneficial effects in individuals undergoing short-term energy deficit where the goal is to optimize body composition without loss of skeletal muscle mass.

[0009] In a general embodiment, a method for enhancing muscle protein synthesis in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein in an amount from about 15 g to about 30 g during a time period selected from the group consisting of during exercise, post-exercise, or combinations thereof.

[0010] In another embodiment, a method for enhancing skeletal muscle translation initiation signaling in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein in an amount from about 15 g to about 30 g during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof.

[0011] In yet another embodiment, a method for enhancing messenger ribonucleic acid (mRNA) expression in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein in an amount from about 15 g to about 30 g during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof.

[0012] In still yet another embodiment, a method for enhancing myofibrillar protein synthesis in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein in an amount from about 15 g to about 30 g during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof.

[0013] In another embodiment, a method for reducing a loss of fat free mass in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein in an amount from about 15 g to about 30 g during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof.

[0014] In yet another embodiment, a method for enhancing muscle protein synthesis in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof, wherein the protein is administered in a dose that is higher than a dose typically recommended for individuals experiencing energy balance.

[0015] In still yet another embodiment, a method for enhancing skeletal muscle translation initiation signaling in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof, wherein the protein is administered in a dose that is higher than a dose typically recommended for individuals experiencing energy balance. [0016] In yet another embodiment, a method for enhancing messenger ribonucleic acid (mRNA) expression in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein during a time period selected from the group consisting of during exercise, post- exercise, and combinations thereof, wherein the protein is administered in a dose that is higher than a dose typically recommended for individuals experiencing energy balance.

[0017] In another embodiment, a method for enhancing myofibrillar protein synthesis in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof, wherein the protein is administered in a dose that is higher than a dose typically recommended for individuals experiencing energy balance.

[0018] In yet another embodiment, a method for reducing a loss of fat free mass in an individual suffering from energy deficit is provided. The method includes administering to the individual a composition comprising protein during a time period selected from the group consisting of during exercise, post-exercise, and combinations thereof, wherein the protein is administered in a dose that is higher than a dose typically recommended for individuals experiencing energy balance.

[0019] In an embodiment, the composition is administered immediately following a resistance exercise.

[0020] In an embodiment, the protein is whey protein. The protein may also be a high-quality protein. In this regard, the protein may have a PDCAAS of greater than 0.7, or greater than 0.8, or greater than 0.9, or the like. Alternatively, high quality protein can be mixed with a lower quality protein wherein the resulting PDCAAS of the mixture is 0.65 or greater, or 0.7 or greater, or the like.

[0021] In an embodiment, the individual is suffering from a short-term energy deficit. The short-term energy deficit may range from about 1 day to about 10 days, or from about 2 days to about 9 days, or from about 3 days to about 8 days, or from about 4 days to about 7 days, or from about 5 days to about 6 days. In an embodiment, the short- term energy deficit is about 5 days. [0022] In an embodiment, the composition is formulated for administration to an individual selected from one of an infant, a child, a young adult, an elderly adult, an athlete, an individual undergoing rehabilitation, an individual in need of weight management, an individual suffering from malnutrition, a febrile individual, a sick individual, or combinations thereof.

[0023] In an embodiment, the administration occurs through an administration route selected from the group consisting of orally, a tube, a catheter, or combinations thereof.

[0024] In an embodiment, the composition further comprises a source of ω-3 fatty acids selected from the group consisting of fish oil, krill, plant sources containing co-3 fatty acids, flaxseed, walnut, algae, or combinations thereof. In an embodiment, the ω-3 fatty acids are selected from the group consisting of a-linolenic acid ("ALA"), docosahexaenoic acid ("DHA"), eicosapentaenoic acid ("EPA"), or combinations thereof.

[0025] In an embodiment, the composition further comprises at least one nucleotide selected from the group consisting of a subunit of deoxyribonucleic acid ("DNA"), a subunit of ribonucleic acid ("RNA"), polymeric forms of DNA and RNA, yeast RNA, or combinations thereof. In an embodiment, the at least one nucleotide is an exogenous nucleotide.

[0026] In an embodiment, the composition further comprises a phytonutrient selected from the group consisting of flavanoids, allied phenolic compounds, polyphenolic compounds, terpenoids, alkaloids, sulphur- containing compounds, or combinations thereof.

[0027] In an embodiment, the phytonutrient is selected from the group consisting of carotenoids, plant sterols, quercetin, curcumin, limonin, or combinations thereof.

[0028] In an embodiment, the composition further comprises a prebiotic selected from the group consisting of acacia gum, alpha glucan, arabinogalactans, beta glucan, dextrans, fructooligosaccharides, fucosyllactose, galactooligosaccharides, galactomannans, gentiooligosaccharides, glucooligosaccharides, guar gum, inulin, isomaltooligosaccharides, lactoneotetraose, lactosucrose, lactulose, levan, maltodextrins, milk oligosaccharides, partially hydrolyzed guar gum, pecticoligosaccharides, resistant starches, retrograded starch, sialooligosaccharides, sialyllactose, soyoligosaccharides, sugar alcohols, xylooligosaccharides, their hydrolysates, or combinations thereof.

[0029] In an embodiment, the composition further comprises a probiotic selected from the group consisting of Aerococcus, Aspergillus, Bacteroides, Bifidobacterium, Candida, Clostridium, Debaromyces, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, Mucor, Oenococcus, Pediococcus, Penicillium, Peptostrepococcus, Pichia, Propionibacterium, Pseudocatenulatum, Rhizopus, Saccharomyces, Staphylococcus, Streptococcus, Torulopsis, Weissella, non-replicating microorganisms, or combinations thereof.

[0030] In an embodiment, the composition further comprises an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, hydroxyserine, hydroxytyrosine, hydroxylysine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine, valine, or combinations thereof.

[0031] In an embodiment, the composition further comprises an antioxidant selected from the group consisting of astaxanthin, carotenoids, coenzyme Q10 ("CoQIO"), flavonoids, glutathione, Goji (wolfberry), hesperidin, lactowolfberry, lignan, lutein, lycopene, polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, or combinations thereof.

[0032] In an embodiment, the composition further comprises a vitamin selected from the group consisting of vitamin A, Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folic acid), and Vitamin B12 (various cobalamins; commonly cyanocobalamin in vitamin supplements) _, vitamin C, vitamin D, vitamin E, vitamin K, Kl and K2 (i.e., MK-4, MK-7), folic acid, biotin, or combinations thereof.

[0033] In an embodiment, the composition further comprises a mineral selected from the group consisting of boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or combinations thereof. [0034] In an embodiment, the composition is an oral nutritional supplement. Alternatively, the nutritional composition may be a tube feeding.

[0035] In an embodiment, the composition is a source of complete nutrition. Alternatively, the nutritional composition may be a source of incomplete nutrition.

[0036] An advantage of the present disclosure is to provide methods for enhancing muscle protein synthesis.

[0037] Yet another advantage of the present disclosure is to provide methods for enhancing muscle protein synthesis.

[0038] Another advantage of the present disclosure is to provide methods for enhancing muscle anabolism.

[0039] Yet another advantage of the present disclosure is to demonstrate the impact of exercise and post-exercise protein intake on muscle protein synthesis during periods of energy deficit.

[0040] Another advantage of the present disclosure is to demonstrate the impact of exercise and post-exercise protein intake on skeletal muscle translation initiation signaling, mRNA expression, and rates of muscle protein synthesis during periods of energy deficit.

[0041] Still yet another advantage of the present disclosure is to provide methods for enhancing physical rehabilitation.

[0042] Yet another advantage of the present disclosure is to provide methods for muscle recovery after exercise.

[0043] Additional features and advantages are described herein, and will be apparent from the following Detailed Description and the figures.

BRIEF DESCRIPTION OF THE FIGURES

[0044] FIG. 1 shows a schematic of an experimental design in accordance with an embodiment of the present disclosure. The resting energy balance (EB) trial was preceded by five (5) days of controlled diet providing 45 kcal · kg ^_1 FFM · day. The resting/exercise in energy deficit (ED) trials were preceded by five (5) days of controlled diet providing 30 kcal · kg ^_1 FFM · day. Solid arrow, muscle biopsy sample; *, blood sample; REX, resistance exercise; 0 g, 15 g and 30 g represent the respective placebo or whey protein drinks (500 mL). Dashed time-line represent trials undertaken a single time by each subject. Times in parentheses are for ED trials involving exercise and protein intake.

[0045] FIG. 2 shows plasma insulin concentration after five (5) days of energy deficit (30 kcal · kg ^_1 FFM · day ) following a bout of leg press (6 sets x 8 repetitions at 80% one repetition maximum) and post-exercise ingestion of a placebo (PL) or 15 or 30g of whey protein drinks. Data were analyzed by two way repeated measures ANOVA with Student-Newman-Keuls post hoc analysis. Values are mean ± SD. Different vs a, rest within treatment; *, PL ,†, 15g; §, 30 g at equivalent time point (P < 0.05).

[0046] FIG. 3 shows (A) plasma essential amino acids (EAA), (B) branched- chain amino acids (BCAA), and (C) leucine concentration after five (5) days of energy deficit (30 kcal · kg ^_1 FFM · day) and following a bout of leg press (6 sets x 8 repetitions at 80% one repetition maximum) and post-exercise ingestion of a placebo (PL), 15 or 30 g of whey protein drinks. Data were analyzed by using two way repeated measures ANOVA with Student-Newman-Keuls post hoc analysis. Values are mean ± SD. Different vs. a, rest within treatment;†, 15 g; §, 30 g at equivalent time point (P < 0.05).

[0047] FIG. 4 shows myofibrillar fractional synthetic rate (FSR) at rest after five (5) days of energy balance (45 kcal · kg ^_1 FFM · day; EB), after five (5) days of energy deficit (30 kcal · kg ^_1 FFM · day; ED) and following a bout of leg press (6 sets x 8 repetitions at 80% one repetition maximum) and post-exercise ingestion of a placebo (PL), 15 or 30 g of whey protein drinks. Data were analyzed by using repeated measures ANOVA with Student-Newman-Keuls post hoc analysis. Values are mean ± SD. Different vs. a, EB; b, ED; c, PL; d, 15 g (P < 0.02).

[0048] FIG. 5 shows myofibrillar fractional synthetic rates (FSR) after five (5) days of energy deficit (30 kcal · kg ^_1 FFM · day) following bout of leg press (6 sets x 8 repetitions at 80% one repetition maximum) plotted against post exercise protein intake in grams of protein per kg of (A) body mass (BM) and (B) fat free mass (FFM). Data were analyzed using linear regression. [0049] FIG. 6 shows phosphorylation of (A) skeletal muscle Akt ^ber4 , (B) mTOR ^Ser2448 , (C) p70 S6K ^Thr389 , and (D) rpS6 ^Ser235/236 at rest after five (5) days of energy balance (45 kcal · kg ^_1 FFM · day; EB), after five (5) days of energy deficit (30 kcal · kg ^_1 FFM · day; ED) and following a bout of leg press (6 sets x 8 repetitions at 80% one repetition maximum) and post-exercise ingestion of a placebo (PL), 15 or 30g of whey protein drinks. Data were analyzed by repeated measures ANOVA with Student-Newman- Keuls post hoc analysis. Values are mean ± SD. Different vs. (a) EB; (b) ED; (c) PL 1.5 h; (d) PL 4.5 h; (f) 15 g, 4.5 h; (h) 30 g, 4.5 hour (P < 0.05).

[0050] FIG. 7 shows (A) MuRF-1, (B) Atrogin-1, (C) SLC38A2/SNAT 2, and (D) SLC7A5/LAT1 mRNA abundance at rest after give (5) days of energy balance (45 kcal · kg -1 FFM · day; EB), after five (5) days of energy deficit (30 kcal · kg ^_1 FFM · day; ED) and following a bout of leg press (6 sets x 8 repetitions at 80% one repetition maximum) and post-exercise ingestion of a placebo (PL), 15 or 30 g of whey protein drinks. Data were analyzed by repeated measures ANOVA with Student-Newman-Keuls post hoc analysis. Values are mean ± SD. Different vs. (a) EB; (b) ED; (c) PL 1.5h; (d) PL 4.5 h; (e) 15g, 1.5 g; (g) 30 g, 1.5 hour (P < 0.05).

DETAILED DESCRIPTION

[0051] All dosage ranges contained within this application are intended to include all numbers, whole or fractions, contained within said range.

[0052] As used in this disclosure and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a mixture of two or more polypeptides, and the like.

[0053] As used herein, "about" is understood to refer to numbers in a range of numerals. Moreover, all numerical ranges herein should be understood to include all integer, whole or fractions, within the range.

[0054] As used herein the term "amino acid" is understood to include one or more amino acids. The amino acid can be, for example, alanine, arginine, asparagine, aspartate, citrulline, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, hydroxyserine, hydroxytyrosine, hydroxylysine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine, valine, or combinations thereof.

[0055] As used herein, "animal" includes, but is not limited to, mammals, which include but is not limited to, rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms "animal" or "mammal" or their plurals are used, it is contemplated that it also applies to any animals that are capable of the effect exhibited or intended to be exhibited by the context of the passage.

[0056] As used herein, the term "antioxidant" is understood to include any one or more of various substances such as beta-carotene (a vitamin A precursor), vitamin C, vitamin E, and selenium) that inhibit oxidation or reactions promoted by Reactive Oxygen Species ("ROS") and other radical and non-radical species. Additionally, antioxidants are molecules capable of slowing or preventing the oxidation of other molecules. Non- limiting examples of antioxidants include astaxanthin, carotenoids, coenzyme Q10 ("CoQIO"), flavonoids, glutathione, Goji (wolfberry), hesperidin, lactowolfberry, lignan, lutein, lycopene, polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, or combinations thereof.

[0057] As used herein, "complete nutrition" includes nutritional products and compositions that contain sufficient types and levels of macronutrients (protein, fats and carbohydrates) and micronutrients to be sufficient to be a sole source of nutrition for the animal to which it is being administered to. Patients can receive 100% of their nutritional requirements from such complete nutritional compositions.

[0058] As used herein, "effective amount" is an amount that prevents a deficiency, treats a disease or medical condition in an individual or, more generally, reduces symptoms, manages progression of the diseases or provides a nutritional, physiological, or medical benefit to the individual. A treatment can be patient- or doctor- related. [0059] While the terms "individual" and "patient" are often used herein to refer to a human, the invention is not so limited. Accordingly, the terms "individual" and "patient" refer to any animal, mammal or human having or at risk for a medical condition that can benefit from the treatment.

[0060] As used herein, sources of co-3 fatty acids include, for example, fish oil, krill, plant sources of co-3, flaxseed, walnut, and algae. Examples of co-3 fatty acids include, for example, a-linolenic acid ("ALA"), docosahexaenoic acid ("DHA"), eicosapentaenoic acid ("EPA"), or combinations thereof.

[0061] As used herein, "food grade micro-organisms" means micro- organisms that are used and generally regarded as safe for use in food.

[0062] As used herein, "high-quality protein" refers to protein that has a specific Protein Digestibility Corrected Amino Acid Score (PDCAAS). The PDCAAS is a method of evaluating the quality of protein based on both the amino acid requirements of humans and their ability to digest it. Indeed, the quality of protein depends on the level at which it provides the nutritional amounts of essential amino acids needed for overall body health, maintenance, and growth. For example, animal proteins, such as eggs, cheese, milk, meat, and fish, are considered complete proteins because they provide sufficient amounts of the essential amino acids. Plant proteins, such as grain, corn, nuts, vegetables and fruits, are considered incomplete proteins because many plant proteins lack one or more of the essential amino acids, or because they lack a proper balance of amino acids. Incomplete proteins can, however, be combined to provide all the essential amino acids, though combinations of incomplete proteins must be consumed at the same time, or within a short period of time (within four hours), to obtain the maximum nutritive value from the amino acids. Such combination diets generally yield a high-quality protein meal, providing sufficient amounts and proper balance of the essential amino acids needed by the body to function.

[0063] There are 20 primary amino acids, which include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, seronine, threonine, tryptophan, tyrosine, and valine. The human body, however, is unable to make 9 of these amino acids on its own, so these amino acids are considered "essential" and must come from our diet. The amino acids regarded as essential for humans are phenylalanine, valine, threonine, tryptophan, isoieucme, methionine, leucine, lysine, and histidine. Additionally, cysteine (or sulphur-containing amino acids), tyrosine (or aromatic amino acids), and argmine are required by infants and growing children.

[0064] A protein's quality is determined by investigating whether all 9 essential amino acids are present in the protein and whether the ratios of essential amino acids in the protein are ideal. With respect to the presence of the 9 essential amino acids, pretty much every source of protein has at least some of all the essential amino acids. However, if the protein is deficient in 1 or more essential amino acid then it is considered incomplete. As a point of reference, grains are typically low in the amino acid lysine, while legumes are low in methionine. Animal products, on the other hand, are high in all the essential amino acids and are usually considered complete. Regarding whether the ratios of essential amino acids in the protein are ideal, basically, the body needs a certain proportion of essential amino acids in the protein in order for it to be used most effectively. If all 9 are present yet 1 or more are not very abundant then the protein is of lower quality because the body will not be able to use the protein to its fullest potential.

[0065] A PDCAAS value of 1 is the highest quality protein, and a value of 0 is the lowest quality protein. As used herein, high quality protein is be defined as a PDCAAS of 0.7 or greater, or 0.8 or greater, or 0.9 of greater, or the like. In an embodiment, however, high quality protein may be mixed with a lower quality protein wherein the resulting PDCAAS of the protein mixture is 0.65 or greater, or 0.7 or greater, or the like.

[0066] As used herein, "incomplete nutrition" includes nutritional products or compositions that do not contain sufficient levels of macronutrients (protein, fats and carbohydrates) or micronutrients to be sufficient to be a sole source of nutrition for the animal to which it is being administered to. Partial or incomplete nutritional compositions can be used as a nutritional supplement. [0067] As used herein, "long term administrations" are preferably continuous administrations for more than 6 weeks. Alternatively, "short term administrations," as used herein, are continuous administrations for less than 6 weeks.

[0068] As used herein, "mammal" includes, but is not limited to, rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the term "mammal" is used, it is contemplated that it also applies to other animals that are capable of the effect exhibited or intended to be exhibited by the mammal.

[0069] The term "microorganism" is meant to include the bacterium, yeast and/or fungi, a cell growth medium with the microorganism, or a cell growth medium in which microorganism was cultivated.

[0070] As used herein, the term "minerals" is understood to include boron, calcium, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or combinations thereof.

[0071] As used herein, a "non-replicating" microorganism means that no viable cells and/or colony forming units can be detected by classical plating methods. Such classical plating methods are summarized in the microbiology book: James Monroe Jay, et al., Modern food microbiology, 7th edition, Springer Science, New York, N. Y. p. 790 (2005). Typically, the absence of viable cells can be shown as follows: no visible colony on agar plates or no increasing turbidity in liquid growth medium after inoculation with different concentrations of bacterial preparations ('non replicating' samples) and incubation under appropriate conditions (aerobic and/or anaerobic atmosphere for at least 24h). For example, bifidobacteria such as Bifidobacterium longum, Bifidobacterium lactis and Bifidobacterium breve or lactobacilli, such as Lactobacillus paracasei or Lactobacillus rhamnosus, may be rendered non-replicating by heat treatment, in particular low temperature/long time heat treatment.

[0072] As used herein, a "nucleotide" is understood to be a subunit of deoxyribonucleic acid ("DNA"), ribonucleic acid ("RNA"), polymeric RNA, polymeric DNA, or combinations thereof. It is an organic compound made up of a nitrogenous base, a phosphate molecule, and a sugar molecule (deoxyribose in DNA and ribose in RNA). Individual nucleotide monomers (single units) are linked together to form polymers, or long chains. Exogenous nucleotides are specifically provided by dietary supplementation. The exogenous nucleotide can be in a monomeric form such as, for example, 5'-Adenosine Monophosphate ("5'-AMP"), 5'-Guanosine Monophosphate ("5'-GMP"), 5'-Cytosine Monophosphate ("5'-CMP"), 5'-Uracil Monophosphate ("5'-UMP"), 5'-Inosine Monophosphate ("5'-IMP"), 5'-Thymine Monophosphate ("5'-TMP"), or combinations thereof. The exogenous nucleotide can also be in a polymeric form such as, for example, an intact RNA. There can be multiple sources of the polymeric form such as, for example, yeast RNA.

[0073] "Nutritional products," or "nutritional compositions," as used herein, are understood to include any number of optional additional ingredients, including conventional food additives (synthetic or natural), for example one or more acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifies, excipient, flavor agent, mineral, osmotic agents, a pharmaceutically acceptable carrier, preservatives, stabilizers, sugar, sweeteners, texturizers, and/or vitamins. The optional ingredients can be added in any suitable amount. The nutritional products or compositions may be a source of complete nutrition or may be a source of incomplete nutrition.

[0074] As used herein the term "patient" is understood to include an animal, especially a mammal, and more especially a human that is receiving or intended to receive treatment, as it is herein defined.

[0075] As used herein, "phytochemicals" or "phytonutrients" are non-nutritive compounds that are found in many foods. Phytochemicals are functional foods that have health benefits beyond basic nutrition, are health promoting compounds that come from plant sources, and may be natural or purified. "Phytochemicals" and "Phytonutrients" refers to any chemical produced by a plant that imparts one or more health benefit on the user. Non-limiting examples of phytochemicals and phytonutrients include those that are:

[0076] i) phenolic compounds which include monophenols (such as, for example, apiole, carnosol, carvacrol, dillapiole, rosemarinol); flavonoids (polyphenols) including flavonols (such as, for example, quercetin, fingerol, kaempferol, myricetin, rutin, isorhamnetin), flavanones (such as, for example, fesperidin, naringenin, silybin, eriodictyol), flavones (such as, for example, apigenin, tangeritin, luteolin), flavan-3-ols (such as, for example, catechins, (+)-catechin, (+)-gallocatechin, (-)-epicatechin, (-)- epigallocatechin, (-)-epigallocatechin gallate (EGCG), (-)-epicatechin 3-gallate, theaflavin, theaflavin-3 -gallate, theaflavin- 3 '-gallate, theaflavin-3,3'-digallate, thearubigins), anthocyanins (flavonals) and anthocyanidins (such as, for example, pelargonidin, peonidin, cyanidin, delphinidin, malvidin, petunidin), isoflavones (phytoestrogens) (such as, for example, daidzein (formononetin), genistein (biochanin A), glycitein), dihydroflavonols, chalcones, coumestans (phytoestrogens), and Coumestrol; Phenolic acids (such as: Ellagic acid, Gallic acid, Tannic acid, Vanillin, curcumin); hydroxycinnamic acids (such as, for example, caffeic acid, chlorogenic acid, cinnamic acid, ferulic acid, coumarin); lignans (phytoestrogens), silymarin, secoisolariciresinol, pinoresinol and lariciresinol); tyrosol esters (such as, for example, tyrosol, hydroxytyrosol, oleocanthal, oleuropein); stilbenoids (such as, for example, resveratrol, pterostilbene, piceatannol) and punicalagins;

[0077] ii) terpenes (isoprenoids) which include carotenoids (tetraterpenoids) including carotenes (such as, for example, a-carotene, β-carotene, γ-carotene, δ-carotene, lycopene, neurosporene, phytofluene, phytoene), and xanthophylls (such as, for example, canthaxanthin, cryptoxanthin, aeaxanthin, astaxanthin, lutein, rubixanthin); monoterpenes (such as, for example, limonene, perillyl alcohol); saponins; lipids including: phytosterols (such as, for example, campesterol, beta sitosterol, gamma sitosterol, stigmasterol), tocopherols (vitamin E), and omega-3, 6, and 9 fatty acids (such as, for example, gamma- linolenic acid); triterpenoid (such as, for example, oleanolic acid, ursolic acid, betulinic acid, moronic acid);

[0078] iii) betalains which include Betacyanins (such as: betanin, isobetanin, probetanin, neobetanin); and betaxanthins (non glycosidic versions) (such as, for example, indicaxanthin, and vulgaxanthin);

[0079] iv) organosulfides, which include, for example, dithiolthiones (isothiocyanates) (such as, for example, sulphoraphane); and thiosulphonates (allium compounds) (such as, for example, allyl methyl trisulfide, and diallyl sulfide), indoles, glucosinolates, which include, for example, indole-3-carbinol; sulforaphane; 3,3'- diindolylmethane; sinigrin; allicin; alliin; allyl isothiocyanate; piperine; syn-propanethial- S-oxide;

[0080] v) protein inhibitors, which include, for example, protease inhibitors;

[0081] vi) other organic acids which include oxalic acid, phytic acid (inositol hexaphosphate); tartaric acid; and anacardic acid; or

[0082] vii) combinations thereof.

[0083] As used herein, a "prebiotic" is a food substance that selectively promotes the growth of beneficial bacteria or inhibits the growth or mucosal adhesion of pathogenic bacteria in the intestines. They are not inactivated in the stomach and/or upper intestine or absorbed in the gastrointestinal tract of the person ingesting them, but they are fermented by the gastrointestinal microflora and/or by probiotics. Prebiotics are, for example, defined by Glenn R. Gibson and Marcel B. Roberfroid, "Dietary Modulation of the Human Colonic Microbiota: Introducing the Concept of Prebiotics," J. Nutr., 125: 1401-1412 (1995). Non-limiting examples of prebiotics include acacia gum, alpha glucan, arabinogalactans, beta glucan, dextrans, fructooligosaccharides, fucosyllactose, galactooligosaccharides, galactomannans, gentiooligosaccharides, glucooligosaccharides, guar gum, inulin, isomaltooligosaccharides, lactoneotetraose, lactosucrose, lactulose, levan, maltodextrins, milk oligosaccharides, partially hydrolyzed guar gum, pecticoligosaccharides, resistant starches, retrograded starch, sialooligosaccharides, sialyllactose, soyoligosaccharides, sugar alcohols, xylooligosaccharides, or their hydrolysates, or combinations thereof.

[0084] As used herein, probiotic micro-organisms (hereinafter "probiotics") are food-grade microorganisms (alive, including semi-viable or weakened, and/or non- replicating), metabolites, microbial cell preparations or components of microbial cells that could confer health benefits on the host when administered in adequate amounts, more specifically, that beneficially affect a host by improving its intestinal microbial balance, leading to effects on the health or well-being of the host. See, Salminen S, Ouwehand A. Benno Y. et al, "Probiotics: how should they be defined?," Trends Food Sci. TechnoL, 10, 107-10 (1999). In general, it is believed that these micro-organisms inhibit or influence the growth and/or metabolism of pathogenic bacteria in the intestinal tract. The probiotics may also activate the immune function of the host. For this reason, there have been many different approaches to include probiotics into food products. Non-limiting examples of probiotics include Aerococcus, Aspergillus, Bacteroides, Bifidobacterium, Candida, Clostridium, Debaromyces, Enterococcus, Fusobacterium, Lactobacillus, Lactococcus, Leuconostoc, Melissococcus, Micrococcus, Mucor, Oenococcus, Pediococcus, Penicillium, Peptostrepococcus, Pichia, Propionibacterium, Pseudocatenulatum, Rhizopus, Saccharomyces, Staphylococcus, Streptococcus, Torulopsis, Weissella, or combinations thereof.

[0085] The terms "protein," "peptide," "oligopeptides" or "polypeptide," as used herein, are understood to refer to any composition that includes, a single amino acids (monomers), two or more amino acids joined together by a peptide bond (dipeptide, tripeptide, or polypeptide), collagen, precursor, homolog, analog, mimetic, salt, prodrug, metabolite, or fragment thereof or combinations thereof. For the sake of clarity, the use of any of the above terms is interchangeable unless otherwise specified. It will be appreciated that polypeptides (or peptides or proteins or oligopeptides) often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes such as glycosylation and other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the known modifications which may be present in polypeptides of the present invention include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavanoid or a heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycation, glycosylation, glycosylphosphatidyl inositol ("GPI") membrane anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to polypeptides such as arginylation, and ubiquitination. The term "protein" also includes "artificial proteins" which refers to linear or non-linear polypeptides, consisting of alternating repeats of a peptide.

[0086] Non-limiting examples of proteins include dairy based proteins, plant based proteins, animal based proteins and artificial proteins. Dairy based proteins may be selected from the group consisting of casein, caseinates, casein hydrolysate, whey, whey hydrolysates, whey concentrates, whey isolates, milk protein concentrate, milk protein isolate, or combinations thereof. Plant based proteins include, for example, soy protein (e.g., all forms including concentrate and isolate), pea protein (e.g., all forms including concentrate and isolate), canola protein (e.g., all forms including concentrate and isolate), other plant proteins that commercially are wheat and fractionated wheat proteins, corn and it fractions including zein, rice, oat, potato, peanut, and any proteins derived from beans, buckwheat, lentils, pulses, single cell proteins, or combinations thereof. Animal based proteins may be selected from the group consisting of beef, poultry, fish, lamb, seafood, or combinations thereof.

[0087] As used herein, the term "rehabilitation" refers to the process of restoring an individual to good, physical condition, operation, or capacity after decrease of same. Accordingly, rehabilitation may include physical therapy, exercise or the like. Examples of individuals in need of rehabilitation include, but are not limited to, individuals having had muscle losses due to immobilization/bed rest, individuals in the hospital, individuals recovering from a critical illness or acute disease, individuals suffering from physical limitations, elderly individuals, athletes, infants experiencing growth retardation, etc.

[0088] As used herein, the terms "treatment," "treat" and "to alleviate" include both prophylactic or preventive treatment (that prevent and/or slow the development of a targeted pathologic condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The term does not necessarily imply that a subject is treated until total recovery. The terms "treatment" and "treat" also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition, such as nitrogen imbalance or muscle loss. The terms "treatment," "treat" and "to alleviate" are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measure. The terms "treatment," "treat" and "to alleviate" are further intended to include the dietary management of a disease or condition or the dietary management for prophylaxis or prevention a disease or condition.

[0089] As used herein, a "tube feed" is a complete or incomplete nutritional product or composition that is administered to an animal's gastrointestinal system, other than through oral administration, including but not limited to a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube ("J-tube"), percutaneous endoscopic gastrostomy ("PEG"), port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.

[0090] As used herein the term "vitamin" is understood to include any of various fat-soluble or water-soluble organic substances (non-limiting examples include vitamin A, Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folic acid), and Vitamin B12 (various cobalamins; commonly cyanocobalamin in vitamin supplements) _, vitamin C, vitamin D, vitamin E, vitamin K, Kl and K2 (i.e. MK-4, MK-7), folic acid and biotin) essential in minute amounts for normal growth and activity of the body and obtained naturally from plant and animal foods or synthetically made, provitamins, derivatives, analogs.

[0091] The present disclosure is related to methods for enhancing muscle protein synthesis. As discussed previously, negative energy balance or energy deficit (ED) can be achieved through reduced energy intake and/or increase energy expenditure and lead to fat mass (FM) loss. A reduction in FM is a common goal for both athletic performance and improved health outcomes (Mettler S, Mitchell N, Tipton KD. Increased protein intake reduces lean body mass loss during weight loss in athletes, Med Sci Sports Exerc 2010;42(2):326-37; Hansen D, Dendale P, Berger J, van Loon LJ, Meeusen R, The effects of exercise training on fat-mass loss in obese patients during energy intake restriction. Sports Med 2007;37(l):31-46). However, when a reduction in fat mass is achieved by energy restriction alone, it typically results in the concomitant loss of fat free mass FFM, predominantly skeletal muscle (Parr EB, Coffey VG, Hawley JA. 'Sarcobesity': a metabolic conundrum. Maturitas 2013;74(2): 109-13). Given that the quality and quantity of skeletal muscle is a major determinant of athletic performance, whole body metabolic rate and functional capacity throughout the life span (Karagounis LG, Hawley JA. Skeletal muscle: increasing the size of the locomotor cell. Int J Biochem Cell Biol 2010;42(9): 1376-9), nutritional and exercise strategies to prevent or minimize loss of FFM while losing fat mass are crucial.

[0092] Pasiakos and colleagues (Pasiakos SM, Vislocky LM, Carbone JW, Altieri N, Konopelski K, Freake HC, Anderson JM, Ferrando AA, Wolfe RR, Rodriguez NR. Acute Energy Deprivation Affects Skeletal Muscle Protein Synthesis and Associated Intracellular Signaling Proteins in Physically Active Adults. J Nutr 2010;140(4):745-51) reported a 19% reduction in basal rates of mixed muscle protein synthesis in young healthy males and females after 10 days of ED (-500 kcal/day). In contrast, a recent study (Pasiakos SM, Cao JJ, Margo s LM, Sauter ER, Whigham LD, McClung JP, Rood JC, Carbone JW, Combs GF, Young AJ. Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial. The FASEB Journal (2013)) from the same group reported no decrease in rates of resting muscle protein synthesis after 30 day of moderate ED. If potential decrease in basal rates of muscle protein synthesis are not accompanied by a concomitant reduction in muscle protein breakdown, then ED could lead to an imbalance in resting protein turnover that could favour the net loss of skeletal muscle proteins. Indeed, prolonged ED-induced body weight loss can be comprised of up to 60% fat free mass (see, id.). In contrast, exercise has been shown to attenuate the loss of lean body mass that can occur with periods of ED. See, Stiegler P, Cunliffe A. The role of diet and exercise for the maintenance of fat-free mass and resting metabolic rate during weight loss. Sports Med 2006;36(3):239-62. However, currently it is unknown if the anabolic effects of resistance exercise are attenuated during periods of ED. Therefore, there is a clear need to further understand the effects of ED on the regulation of muscle protein synthesis.

[0093] Provision of dietary amino acids increases skeletal myofibrillar protein synthesis (MPS), an effect that is enhanced by prior resistance exercise. See, Biolo G, Tipton KD, Klein S, Wolfe RR. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. Am J Physiol 1997;273(1 Pt l):E122-9; Moore DR, Tang JE, Burd NA, Rerecich T, Tarnopolsky MA, Phillips SM. Differential stimulation of myofibrillar and sarcoplasmic protein synthesis with protein ingestion at rest and after resistance exercise. J Physiol 2009;587(4):897-904. Whether skeletal muscle exhibits 'anabolic resistance' to exercise and protein ingestion following short-term ED has not been determined. Moreover, the effects of increased protein availability in this situation are not known. Hence, the primary aim of the study described herein was to determine the effects of resistance exercise and post-exercise protein intake on skeletal muscle translation initiation signaling, mRNA expression and rates of muscle protein synthesis after short-term, moderate energy restriction. In addition, as women may be more susceptible to disregulations of normal metabolism during periods of ED (Loucks AB, Kiens B, Wright HH. Energy availability in athletes. J Sports Sci 2011;29 Suppl 1 :S7- 15), a second aim of the study was to identify potential sex based differences in skeletal muscle anabolism in response to energy deficit.

[0094] Indeed, Applicant surprisingly determined that five days of moderate energy deficit (Energy availability; 30 kcal-kg ^"1 FFM- day ^"1 ) resulted in a reduction (27%) of resting MPS in young, healthy subjects. However, resistance exercise undertaken in a fasted, energy restricted state enhanced MPS but only to basal values in energy balance. Ingestion of protein following resistance exercise undertaken in energy deficit enhanced the anabolic (MPS and signaling) response above resting energy balance the amino acid transporter transcriptional response to resistance exercise and protein ingestion are down regulated in healthy individuals in ED.

[0095] Pasiakos and colleagues have previously reported that 10 days of energy deficit (-500 kcal · day ^"1 ) reduced resting rates of mixed muscle protein synthesis by -19%. See, Pasiakos SM, Vislocky LM, Carbone JW, Altieri N, Konopelski K, Freake HC, Anderson JM, Ferrando AA, Wolfe RR, Rodriguez NR. Acute Energy Deprivation Affects Skeletal Muscle Protein Synthesis and Associated Intracellular Signaling Proteins in Physically Active Adults. J Nutr 2010;140(4):745-51. Applicant's data confirmed the physiological response to energy deficit in vivo skeletal muscle and provided novel data to show there is a similar detrimental 27% decrease on myofibrillar FSR after only 5 days of moderate energy restriction. Applicant chose the model of energy availability to set the energy deficit in our subjects, with a level of 30 kcal-kg ^"1 FFM-day ^"1 corresponding to a de facto threshold below which there is significant disruption to metabolic and hormonal systems within the body. See, Loucks AB, Kiens B, Wright HH. Energy availability in athletes. J Sports Sci 2011;29 Suppl 1 : S7-15. The notional energy deficit of -15 kcal-kg ^"1 FFM-day ^"1 in Applicant's subjects was typically equivalent to 1690-2200 kcal · day ^"1 in the male and 1210-1640 kcal · day ^"1 in the female subjects. To our surprise, the extensive resistance training history of our subjects coupled with the high relative dietary protein intake (1.4 g-kg ^"1 BM-day ^"1 ) during this energy deficit was still unable to "protect" the muscle and preserve resting rates of MPS. Given that protein synthesis is an energetically expensive process, Applicant proposes that the decrease in muscle protein synthesis observed in the present and previous studies (Pasiakos SM, Vislocky LM, Carbone JW, Altieri N, Konopelski K, Freake HC, Anderson JM, Ferrando AA, Wolfe RR, Rodriguez NR. Acute Energy Deprivation Affects Skeletal Muscle Protein Synthesis and Associated Intracellular Signaling Proteins in Physically Active Adults. J Nutr 2010;140(4):745-51) may represent an accommodative physiological response to the reduced energy availability that subsequently down regulates non-essential metabolic pathways.

[0096] Consistent with observations in energy balance (Moore DR, Tang JE, Burd NA, Rerecich T, Tarnopolsky MA, Phillips SM. Differential stimulation of myofibrillar and sarcoplasmic protein synthesis with protein ingestion at rest and after resistance exercise. J Physiol 2009;587(4): 897-904; Phillips SM, Tipton KD, Aarsland A, Wolf SE, Wolfe RR. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. Am J Physiol 1997;273(1 Pt 1):E99-107), the anabolic stimulus generated by resistance exercise in the fasted state elevated MPS above resting levels in the early post-exercise period, during energy restriction. However, despite this elevation, exercise merely "rescued" the rate of MPS in energy restriction to a level that was similar to, but did not exceed, rates measured at rest when in EB. Accordingly, it appears the metabolic status of the muscle during short-term (e.g., 5 days) energy deficit plus an approximately 10 hour fast may dictate that contractile overload in isolation is not enough to increase FSR to values that otherwise would be reached in EB.

[0097] The anabolic effect of protein ingestion is well-accepted. See, Breen L, Phillips SM. Nutrient interaction for optimal protein anabolism in resistance exercise. Curr Opin Clin Nutr Metab Care 2012;15(3):226-32. A recent study on young healthy subjects involving 21 days of high (1.6 and 2.4 g-kg ^"1 BM-day ^"1 ) protein intake while in moderate ED (750 kcal-day ^"1 ) rescued the FFM loss seen with normal (RDA, 0.8 g-kg ^"1 BM) protein intake diet group. Applicant's study described below is the first study to determine the acute muscle anabolic response to different quantities of protein ingested after exercise undertaken during short-term ED. The results from the present study also highlight the importance of resistance exercise together with increasing doses of protein to maximize protein accretion in a chronic intervention. As discussed below, Applicant surprisingly reports that a dose-dependent response MPS to protein ingestion remains evident despite ED as Applicant observed a hierarchical increase above resting energy balance rates of muscle protein synthesis with ingestion of 15 and 30 g whey protein (see, FIG. 4). This effect is evident when protein ingestion is considered in both absolute and relative terms to body mass and fat free mass (see, FIGS. 5). Therefore, exogenous amino acid provision in ED appears to be pre-requisite for supporting muscle protein synthesis that may result in net new muscle protein synthesis above resting energy balance. See, Phillips SM, Tipton KD, Aarsland A, Wolf SE, Wolfe RR. Mixed muscle protein synthesis and breakdown after resistance exercise in humans. Am J Physiol 1997;273(1 Pt 1):E99-107; Biolo G, Maggi SP, Williams BD, Tipton KD, Wolfe RR. Increased rates of muscle protein turnover and amino acid transport after resistance exercise in humans. Am J Physiol 1995;268(3):E514-E20. It is tempting to speculate that the rates of protein synthesis in the present study were inferior to those induced by comparable resistance exercise and post exercise protein ingestion undertaken in energy balance (West DW, Burd NA, Coffey VG, Baker SK, Burke LM, Hawley J A, Moore DR, Stellingwerff T, Phillips SM. Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise. Am J Clin Nutr 2011;94(3):795-803; Areta JL, Burke LM, Ross ML, Camera DM, West DWD, Broad EM, Jeacocke NA, Moore DR, Stellingwerff T, Phillips SM, et al. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis. J Physiol 2013). For example, increases in myofibrillar rates of protein synthesis above rest when employing similar resistance exercise and protein ingestion protocols in energy balance are typically greater than 100% (Moore DR, Tang JE, Burd NA, Rerecich T, Tarnopolsky MA, Phillips SM. Differential stimulation of myofibrillar and sarcoplasmic protein synthesis with protein ingestion at rest and after resistance exercise. J Physiol 2009;587(4):897-904; West DW, Burd NA, Coffey VG, Baker SK, Burke LM, Hawley JA, Moore DR, Stellingwerff T, Phillips SM. Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise. Am J Clin Nutr 2011;94(3):795-803; Areta JL, Burke LM, Ross ML, Camera DM, West DWD, Broad EM, Jeacocke NA, Moore DR, Stellingwerff T, Phillips SM, et al. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis. J Physiol 2013; Moore DR, Robinson MJ, Fry JL, Tang JE, Glover EI, Wilkinson SB, Prior T, Tarnopolsky MA, Phillips SM. Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. Am J Clin Nutr 2009;89(1): 161-8) compared with -60-80% in ED in the current study. Applicant's study is limited to quantification of muscle protein synthesis and the effect of ED on muscle protein breakdown is unknown. Whether resistance exercise and amino acid provision can generate positive net protein balance in skeletal muscle during ED remains to be established; however, there are published examples of individuals concurrently gaining or maintaining LBM while losing total body mass and fat mass in prolonged training studies involving ED (Mettler S, Mitchell N, Tipton KD. Increased protein intake reduces lean body mass loss during weight loss in athletes. Med Sci Sports Exerc 2010;42(2): 326-37; Garthe I, Raastad T, Egil Refsnes P, Koivisto A, Sundgot- Borgen J. Effect of two different weight-loss rates on body composition and strength and power-related performance in elite athletes. International Journal of Sport Nutrition andExercise Metabolism 2011;21(2):97; Haakonssen EC, Martin DT, Burke LM, Jenkins DG. Increased Lean Mass with Reduced Fat Mass in an Elite Female Cyclist Returning to Competition: Case Study. Int J Sports Physiol Perform 2013).

[0098] In the present study, Applicant found no sex-based differences for any of the cellular markers of 'muscle anabolism' measured, providing further support for the notion that both acute and chronic responses to resistance exercise and/or protein ingestion are similar between younger men and women (West DWD, Burd NA, Churchward- Venne TA, Camera DM, Mitchell CJ, Baker SK, Hawley J A, Coffey VG, Phillips SM. Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state. J Appl Physiol 2012;112(11): 1805-13; Phillips BE, Williams JP, Gustafsson T, Bouchard C, Rankinen T, Knudsen S, Smith K, Timmons JA, Atherton PJ. Molecular networks of human muscle adaptation to exercise and age. PLoS Genet 2013;9(3); Smith GI, Mittendorfer B. Similar muscle protein synthesis rates in young men and women: men aren't from Mars and women aren't from Venus. J Appl Physiol 2012;112(11); Smith G, Reeds D, Hall A, Chambers K, Finck B, Mittendorfer B. Sexually dimorphic effect of aging on skeletal muscle protein synthesis. Biology of Sex Differences 2012;3(1): 11.

[0099] The muscle anabolic responses in ED that Applicant found were evident despite differences in body mass and body composition (see, Table 1 below). However, there was a moderate relationship between the relative quantity of protein ingested and the muscle fractional synthetic rate such that Applicant cannot rule out that the (smaller) females may have benefited, at least in part, from a greater relative protein dose (see, FIGS. 5A-B). Whether a greater relative effect on muscle protein synthesis in females would ameliorate any potential sex-based difference in ED seems unlikely. Indeed, Phillips and co-workers (Phillips BE, Williams JP, Gustafsson T, Bouchard C, Rankinen T, Knudsen S, Smith K, Timmons JA, Atherton PJ. Molecular networks of human muscle adaptation to exercise and age. PLoS Genet 2013 ;9(3): el 003389) have recently reported the capacity of skeletal muscle to hypertrophy during 20-week resistance training program as being, to a large extent, genetically determined, is not sex-dependent. Regardless, Applicant's data indicates that the physiological response in skeletal muscle following the short-term ED protocol employed in the current study was similar in male and female subjects and they appear equally responsive to an acute bout of REX and post exercise protein intake in ED.

[00100] TABLE 1. - Subjects characteristics. 1 Repetition Maximum (1 RM); Body Mass (BM); Fat Free Mass (FFM). Data was analyzed by using multiple T-tests. Values are mean ± SD.* different between sexes (P < 0.05).

Males Females

N 8 7

Age (Years) 27 ± 5 28 ± 4

Body mass (kg) * 82.7 ± 6.6 70.3 ± 7

Fat (% BM) * 14.9 ± 3.5 28.7 ± 5.3

Lean Mass (kg) * 67.2 ± 6.2 48.1 ± 4.3

1 RM (kg) * 300 ± 70 200 ± 38

1 RM (kg)/BM (kg) * 3.6 ± 0.8 2.8 ± 0.3

1 RM (kg)/ FFM (kg) 4.5 ± 0.9 4.2 ± 0.7

[00101] The mTOR associated translational signaling data found by Applicant did not reveal any differences at rest between EB and ED, which are in agreement with recent observations by others (Pasiakos SM, Cao JJ, Margolis LM, Sauter ER, Whigham LD, McClung JP, Rood JC, Carbone JW, Combs GF, Young AJ. Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial. The FASEB Journal 2013). As shown below, in the present study, resistance exercise alone undertaken in energy deficit had little effect in promoting the phosphorylation of any of the proteins of interest. This finding is in contrast to Applicant's previous study (Camera DM, Edge J, Short MJ, Hawley JA, Coffey VG. Early time course of Akt phosphorylation after endurance and resistance exercise. Med Sci Sports Exerc 2010;42(10): 1843-52) and the results from several other groups (Creer A, Gallagher P, Slivka D, Jemiolo B, Fink W, Trappe S. Influence of muscle glycogen availability on ERKl/2 and Akt signaling after resistance exercise in human skeletal muscle. J Appl Physiol 2005;99(3): 950-6; Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E, Rasmussen BB. Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J Physiol 2006;576(2):613-24) when resistance exercise is undertaken in the fasted state in EB. However, Applicant did observe a marked increase in translational signaling following the post exercise ingestion of protein, albeit with only subtle differences between the response to 15 g and 30 g of whey protein. Moreover, Applicant has previously shown a hierarchical signaling response to increasing quantities of whey protein ingestion i.e., more protein ingested resulted in greater phosphorylation of p70 S6K (Areta JL, Burke LM, Ross ML, Camera DM, West DWD, Broad EM, Jeacocke NA, Moore DR, Stellmgwerff T, Phillips SM, et al. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis. J Physiol 2013); the results of the current study indicate that energy deficit may alter the magnitude of signal for translation initiation in response to acute exercise and protein intake (see, FIGS. 6A-D). Importantly, the similar phosphorylation responses were not mirrored by myofibrillar FSR and support previous work showing translation initiation signaling can be indicative of elevated muscle protein synthesis compared to baseline (Burd NA, Holwerda AM, Selby KC, West DWD, Staples AW, Cain NE, Cashaback JGA, Potvin JR, Baker SK, Phillips SM. Resistance exercise volume affects myofibrillar protein synthesis and anabolic signalling molecule phosphorylation in young men. J Physiol 2010;588(16):3119-30; Kumar V, Atherton P, Smith K, Rennie MJ. Human muscle protein synthesis and breakdown during and after exercise. J Appl Physiol 2009;106(6):2026-39; Fry CS, Drummond MJ, Glynn EL, Dickinson JM, Gundermann DM, Timmerman KL, Walker DK, Dhanani S, Volpi E, Rasmussen BB. Aging impairs contraction-induced human skeletal muscle mTORCl signaling and protein synthesis. Skelet Muscle 2011 ;1(1): 11), but does not accurately reflect the magnitude or duration of the muscle protein synthesis response (Areta JL, Burke LM, Ross ML, Camera DM, West DWD, Broad EM, Jeacocke NA, Moore DR, Stellingwerff T, Phillips SM, et al. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis. J Physiol 2013). [00102] As shown below, energy deficit did not generate any differences in muscle transcriptional activity of any gene of interest at rest compared with resting energy balance (see, FIGS. 7A-D). The ubiquitin ligases MuRF-1, and Atrogin are key regulatory steps of the ubiquitin-proteasomal protein degradation. Originally linked to muscle atrophy they seem to be important in the myofibril remodelling process after a bout of resistance exercise. The mRNA abundance of MuRF-1 increased above rest and placebo following post exercise ingestion of 15 g protein, but was not different to the higher (30 g) protein dose. Protein intake in sufficient quantities has been previously shown to blunt the exercise induced increase in MuRF-1 mRNA abundance (Areta JL, Burke LM, Ross ML, Camera DM, West DWD, Broad EM, Jeacocke NA, Moore DR, Stellmgwerff T, Phillips SM, et al. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis. J Physiol 2013; Borgenvik M, Apro W, Blomstrand E. Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle. Am J Physiol 2012;302(5):E510-E21; Louis E, Raue U, Yang Y, Jemiolo B, Trappe S. Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle. J Appl Physiol 2007;103(5): 1744-51). Interestingly, increases in Atrogin mRNA abundance following high intensity exercise is not consistently observed in vivo humans (Areta JL, Burke LM, Ross ML, Camera DM, West DWD, Broad EM, Jeacocke NA, Moore DR, Stellingwerff T, Phillips SM, et al. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis. J Physiol 2013; Louis E, Raue U, Yang Y, Jemiolo B, Trappe S. Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle. J Appl Physiol 2007; 103(5): 1744-51 ; Raue U, Slivka D, Jemiolo B, Hollon C, Trappe S. Proteolytic gene expression differs at rest and after resistance exercise between young and old women. J Gerontol A Biol Sci Med Sci 2007;62(12): 1407-12) and Applicant's results showing elevated Atrogin mRNA following resistance exercise in all treatments indicates energy deficit may promote the catabolic activity of this specific atrogene. Importantly, protein ingestion did not alter the elevated transcriptional activity of Atrogin during recovery from resistance exercise in energy deficit but direct measures of protein breakdown are required to determine the physiological relevance of the increase in Atrogin mRNA expression when exercising in ED.

[00103] The results from previous studies have shown resistance exercise and protein feeding augments amino acid transporter mRNA and protein abundance in skeletal muscle (Areta JL, Burke LM, Ross ML, Camera DM, West DWD, Broad EM, Jeacocke NA, Moore DR, Stellingwerff T, Phillips SM, et al. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis. J Physiol 2013; Drummond MJ, Fry CS, Glynn EL, Timmerman KL, Dickinson JM, Walker DK, Gundermann DM, Volpi E, Rasmussen BB. Skeletal muscle amino acid transporter expression is increased in young and older adults following resistance exercise. J Appl Physiol 2011;111(1): 135-42; Drummond MJ, Glynn EL, Fry CS, Timmerman KL, Volpi E, Rasmussen BB. An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle. Am J Physiol 2010;298(5):E1011-E8). Applicant's results, as summarized below, the first in examination of the transport proteins in ED, show either little effect or, indeed, a down regulation of SNAT2 and LAT1 mRNA, respectively, indicative of a suppression of global amino acid transporter expression following exercise and protein ingestion when in ED. Given that exercise and protein intake increased MPS compared to resting ED there does not appear to be any acute relationship between the transcriptional activities of amino acid transporters such that it would prevent any anabolic response. The physiological significance of changes in amino acid transporter mRNA expression are beyond the scope of Applicant's present study, however one could speculate that the down regulation of transporter expression could potentially result in depressing the capacity of amino acid uptake in to skeletal muscle when in chronic energy deficit and represents an area for future study. This phenomenon may be consequence of a physiological adjustment to lower energy availability. Given that turnover is a high energy demanding process, shifting the system towards a state of reduced protein turnover would increase energy availability for processes of higher priority for survival. Alternatively, exercise has been shown to enhance amino acid sensitivity of skeletal muscle to circulating amino acids, which could suggest that the down regulation of transporter mRNA expression with exercise (relative to a fasting control) is a reflection of a reduced transporter requirement to facilitate amino acid uptake. Therefore, the physiological significance of changes in amino acid transporter mRNA and, ultimately, protein expression with exercise and dietary manipulations is further warranted.

[00104] In conclusion, Applicant's studies provide novel data on the effect of a short-term energy restricted diet on skeletal muscle metabolism in vivo humans (Mettler S, Mitchell N, Tipton KD. Increased protein intake reduces lean body mass loss during weight loss in athletes. Med Sci Sports Exerc 2010;42(2): 326-37; Campbell WW, Haub MD, Wolfe RR, Ferrando AA, Sullivan DH, Apolzan JW, Iglay HB. Resistance Training Preserves Fat-free Mass Without Impacting Changes in Protein Metabolism After Weight Loss in Older Women. Obesity 2009; 17(7): 1332-9; Pikosky MA, Smith TJ, Grediagin A, Castaneda-Sceppa C, Byerley L, Glickman EL, Young AJ. Increased protein maintains nitrogen balance during exercise-induced energy deficit. Med Sci Sports Exerc 2008;40(3): 505-12; Layman DK, Evans E, Baum JI, Seyler J, Erickson DJ, Boileau RA. Dietary Protein and Exercise Have Additive Effects on Body Composition during Weight Loss in Adult Women. J Nutr 2005; 135(8): 1903- 10; Walberg JL, Leidy MK, Sturgill DJ, Hinkle DE, Ritchey SJ, Sebolt DR. Macronutrient Content of a Hypoenergy Diet Affects Nitrogen Retention and Muscle Function in Weight Lifters. Int J Sports Med 1988;09(04):261-6). Collectively, Applicant's data shows that 5 days of moderate energy deficit, at an energy availability equivalent to 30 kcal-kg ^"1 FFM-day ^"1 is sufficient to inhibit rates of muscle protein synthesis. Without being bound to any theories, Applicant believes that if such an energy deficit was sustained for prolonged (i.e., several months) periods, it would lead to substantial reductions in muscle mass. Applicant's results also show that targeted use of a bolus of high quality protein ingestion (15-30g) may have the capacity to off-set the muscle catabolic state. Moreover, our data suggest that the dose required to maximally stimulate muscle protein synthesis under energy balance (-20-25 g of protein; see, Moore DR, Robinson MJ, Fry JL, Tang JE, Glover EI, Wilkinson SB, Prior T, Tarnopolsky MA, Phillips SM. Ingested protein dose response of muscle and albumin protein synthesis after resistance exercise in young men. Am J Clin Nutr 2009;89(1): 161-8; Cuthbertson D, Smith K, Babraj J, Leese G, Waddell T, Atherton P, Wackerhage H, Taylor PM, Rennie MJ. Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle. FASEB J 2005;19(3):422-4) may well be higher in individuals in energy deficit. Regardless, the current investigation provides important new information on the effect of energy deficit on skeletal muscle metabolism and shows the attenuation of muscle protein synthesis at rest in energy deficit can be rescued/augmented by the combined anabolic effects of resistance exercise and protein ingestion.

[00105] By way of example and not limitation, the following examples are illustrative of various embodiments of the present disclosure. The formulations and processes below are provided for exemplification only, and they can be modified by the skilled artisan to the necessary extent, depending on the special features that are desired.

[00106] EXAMPLE 1

[00107] Subjects and Methods

[00108] Ethical Approval.

[00109] Subjects were informed of any potential risks involved in the study before providing their written informed consent. The study was approved by the Australian Institute of Sport Ethics Committee and conformed to the standards set by the latest revision of the Declaration of Helsinki.

[00110] Subjects.

[00111] Sixteen young, healthy, resistance trained subjects (8 females, 8 males) were recruited, one failed to finish. Therefore, the data represents that from the remaining 15 participants (see, Table 1 above). Body composition was measured 1-2 weeks before the first experimental trial using a whole body scan narrowed fan-beam dual energy X-ray analysis (DXA Lunar Prodigy, GE Healthcare, Madison, WI) with GE Encore 13.60 software (GE, Madison, WI).

[00112] Experimental Design.

[00113] The study employed a within subject design, with subjects completing four experimental interventions: energy balance (EB) at rest; energy deficit (ED) at rest; and then ED with exercise both with and without protein feeding. All trials were performed in a randomized order with the exception of the EB trial, which was undertaken first (see, FIG. 1).

[00114] Dietary Intervention.

[00115] Subjects were each provided with individualized pre-packaged meals for 5 days before each experimental trial. Before the resting EB trial, subjects were provided with meals equivalent to an energy availability (EA) of 45 kcal-kg ^"1 FFM · day ^"1 , where EA is defined as energy intake minus the energy cost of exercise. For all ED trials, diets consisted of an energy availability of 30 kcal-kg ^"1 FFM -day ^"1 . Between days 1-3 of the dietary control period subjects were permitted to exercise and the diet adjusted to account for the energy expenditure of the exercise sessions and thus restore EA to the set level. The total protein content was 1.4 g-kg ^"1 BM- day ^"1 for both dietary treatments. No alcohol was consumed by the subjects during the 5-day dietary control period, and they refrained from caffeine intake 24 hours before each trial day. Between experimental trials, there was a 9 day 'washout' period during which subjects were encouraged to continue their normal exercise and dietary habits.

[00116] Experimental Trials.

[00117] After 5 days of dietary control, subjects reported to the laboratory between 0700 and 0800 hours after a 10-12 hour overnight fast and a catheter was inserted in the antecubital vein of each arm for blood sampling and tracer infusion. A first (baseline) blood sample was drawn for the resting EB trial (or muscle biopsy from the vastus lateralis was obtained for the ED trials) immediately before a primed, continuous (0.05 pmol-kg^-min ^"1 ; 2 junol-kg ^"1 prime) infusion of -[ring- ¹³ C ₆ ] phenylalanine commenced.

[00118] After a resting period of 3 hours, a muscle biopsy was obtained. For the three non-tracer naive subjects the first muscle biopsy was taken before the infusion started for their EB trial. [00119] The ED trials were undertaken after the resting EB trial with the protein/placebo ingestion randomized and counterbalanced. Drinks contained 15 g or 30 g of protein (IS08 WPI; 86.8 g protein, 1.5 g fat, 3.1 g carbohydrates per 100 g) or no protein in form of a flavour and volume matched placebo drink. Each protein drink was enriched with 5% L-[n ^' «g- ¹³ C6]phenylalanine and mixed with water to a total volume of 500 mL. The first ED trial for each subject was divided in two periods. The first (pre- exercise) period of the trial determined resting ED and was identical to the EB trial with the exception of an initial muscle biopsy. In the second period, subjects undertook a bout of resistance exercise (REX; described subsequently) with muscle biopsies obtained after 1 hour and 4 hours post-exercise recovery. Drinks were ingested immediately following cessation of the exercise bout. The remaining ED trials were identical but primed constant infusion of tracer commenced prior to exercise.

[00120] Exercise.

[00121] One repetition maximum (1 RM) inclined (45°) leg press (GLPH1100, Body-Solid, Forest Park IL) test was completed by each subject a minimum of one week prior to the experimental trials. After a warm up of 2 sets of 5 moderate intensity repetitions the 1 RM was determined as the highest successfully lifted weight during a maximum of 6 attempts. On the day of an ED experimental trial subjects completed 2 warm up sets of 5 repetitions at -50 and -60% 1 RM with 2 minutes rest between sets. The resistance exercise training bout incorporated 6 sets of 8 repetitions at -80% 1 RM with 3 minutes rest between sets. Exercise range of motion was -85° for the knee joint, with leg extension endpoint set at -5° from full extension.

[00122] Biological Samples.

[00123] Blood samples (4 mL) were taken at rest, before the exercise bout and at repeated time-points throughout recovery (see, FIG. 1). Muscle biopsy samples were taken from different incisions, separated by -1 cm using 5 mm Bergstrom needles adapted for manual suction. Muscle was cleaned with saline solution to remove excess blood and immediately frozen in liquid N ₂ . Muscle and plasma samples were stored at -80 °C until subsequent analysis.

[00124] Analytical Procedures

[00125] Insulin and Amino Acid Concentration.

[00126] Plasma insulin concentration was measured using an automated enzyme amplified chemiluminescence Immulite® 1000 system (Siemens diagnostics, Australia) according to manufacturer's guidelines. Plasma amino acids (AA) were analyzed by high performance liquid chromatography to determine amino acid concentrations as described previously (see, West DW, Burd NA, Coffey VG, Baker SK, Burke LM, Hawley JA, Moore DR, Stellingwerff T, Phillips SM. Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise. Am J Clin Nutr 2011;94(3):795-803; Wilkinson SB, Tarnopolsky MA, MacDonald MJ, MacDonald JR, Armstrong D, Phillips SM. Consumption of fluid skim milk promotes greater muscle protein accretion after resistance exercise than does consumption of an isonitrogenous and isoenergetic soy-protein beverage. Am J Clin Nutr 2007;85(4): 1031- 40).

[00127] Western Blot.

[00128] Intracellular signalling proteins were extracted, isolated and quantified as previously described (see, Coffey V, Moore D, Burd N, Rerecich T, Stellingwerff T, Garnham A, Phillips S, Hawley J. Nutrient provision increases signalling and protein synthesis in human skeletal muscle after repeated sprints. Eur J Appl Physiol 2011 ;111(7): 1473-83). Amount of protein loaded in each well was 40 μg. Polyclonal anti- phospho mammalian target of rapamycin (mTOR) Ser2448 (#2971), monoclonal anti- phospho-Akt Ser473 (#9271), nbosomal protein S6 Ser 235/6 (#4856), 4E-BP1 Thr37/46 (# 2855), eEF2 Thr56 (# 2331), AMPK Thrl72 (#2535) and anti-a-tubulin control protein (#3873) were purchased from Cell Signaling Technology (Danvers, MA). Polyclonal anti- phospho- p70S6K Thr 389 (#PK1015) was from Millipore (Temecula, CA). [00129] Fractional Synthetic Rate.

[00130] Pre-infusion plasma sample proteins, extracted by acetonitrile, were utilized as the baseline enrichment in tracer naive subjects (see, Burd NA, Groen BB, Beelen M, Senden JM, Gijsen AP, van Loon LJ. The reliability of using the single-biopsy approach to assess basal muscle protein synthesis rates in vivo in humans. Metabolism 2012;61(7):931-6). For non-tracer naive subjects (n=3, males) a pre-infusion muscle biopsy was used for baseline enrichment. Muscle tissue was processed as previously described (see, Moore DR, Tang JE, Burd NA, Rerecich T, Tarnopolsky MA, Phillips SM. Differential stimulation of myofibrillar and sarcoplasmic protein synthesis with protein ingestion at rest and after resistance exercise. J Physiol 2009;587(4): 897-904).

[00131] Calculations.

[00132] The fractional synthetic rate of myofibrillar proteins was calculated using the standard precursor-product method:

[00133] FSR (%-h ^-1 ) = [E _p2 - E _p i]/E _ic x 1/t x 100

[00134] Where E _p2 - E _p i represents the change in bound protein enrichment between two biopsy samples; Ei _C is the average enrichment of intracellular phenylalanine between the two biopsy samples; and t is the time between biopsies. The utilization of 'tracer-naive' subjects (n=12) allowed us to use the pre-infusion blood sample (i.e. mixed plasma protein fraction) as the baseline enrichment (Epl) for the calculation of resting muscle protein synthesis (Burd NA, Holwerda AM, Selby KC, West DWD, Staples AW, Cain NE, Cashaback JGA, Potvin JR, Baker SK, Phillips SM. Resistance exercise volume affects myofibrillar protein synthesis and anabolic signalling molecule phosphorylation in young men. J Physiol 2010;588(16):3119-30).

[00135] RNA Extraction, Reverse Transcription and RT-PCR.

[00136] Skeletal muscle tissue (-20 mg) was used to isolate RNA using a modification of the acid guanidinium thiocyanate-phenol-chloroform extraction, as previously described (see, Coffey VG, Reeder DW, Lancaster GI, Yeo WK, Febbraio MA, Yaspelkis BB, 3rd, Hawley JA. Effect of high-frequency resistance exercise on adaptive responses in skeletal muscle. Med Sci Sports Exerc 2007;39(12):2135-44). Reverse transcription and real-time Polymerase Chain Reaction (RT-PCR) was performed as previously described (see, Camera DM, West DWD, Burd NA, Phillips SM, Garnham AP, Hawley JA, Coffey VG. Low Muscle Glycogen Concentration Does Not Suppress the Anabolic Response to Resistance Exercise. J Appl Physiol 2012; West DWD, Burd NA, Churchward- Venne TA, Camera DM, Mitchell CJ, Baker SK, Hawley JA, Coffey VG, Phillips SM. Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state. J Appl Physiol 2012;112(11): 1805-13). Taqman-FAM-labelled primer/probes for Atrogin-1 (Hs01041408_ml*), MuRF-1 (Hs00822397_ml *), SLC38A2 (Hs00255854_ml*), and SLC7A5 (Hs00185826_ml) primers (Applied Biosystems, Carlsbad, CA) were used. Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH, HS99999905_ml*) was used as the housekeeping gene. The relative amounts of mRNAs were calculated using the relative quantification (AACT) method (see, Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001 ;25(4):402-8).

[00137] Statistical Analysis.

[00138] Data were analyzed using two way repeated measures analysis of variance (ANOVA) with Student-Newman-Keuls post hoc analysis (gender ^χ time) for cell signaling, RT-PCR and myofibrillar FSR There were no differences between genders and data were subsequently combined for further analysis using one way repeated measures ANOVA with Student-Newman-Keuls post hoc test. Data for plasma insulin and amino acids concentration were analyzed using two way repeated measures ANOVA with Student-Newman-Keuls post hoc test where resting energy balance and energy deficit trials were independently analyzed from the exercise trials. Data for Western blotting were log- transformed prior to analysis. All data are presented as mean ± standard deviation (SD) and the level of statistical significance was set at P < 0.05. [00139] Results

[00140] Plasma Insulin Concentration.

[00141] There were no differences in plasma insulin concentration during the resting EB and ED trials. There was a time x group interaction for plasma insulin concentration (P<0.001) after exercise and protein feeding. Plasma insulin concentration increased above pre-exercise levels only with 15 and 30 g protein ingestion reaching peak values 20-40 minutes post-exercise (see, FIG. 2). Plasma insulin concentration for the 30 g treatment was above resting values from 20 minutes until 2 hours post-exercise (2-4.5 fold P < 0.05), whereas for the 15 g group, plasma insulin concentration was elevated above rest from 20 minutes until 1 hour post-exercise (2-3.5 fold; P < 0.05). Plasma insulin was higher in the 15 and 30 g treatments compared to placebo 20-40 minutes post-exercise (2.3-3.8 fold; P < 0.001), however only 30 g was different compared with PL later at 1 and 2 hours post-exercise (1.7-2 fold; P < 0.05) and was also higher than 15 g at 2 hours post- exercise (1.9 fold ; P < 0.02).

[00142] Amino Acids Concentration.

[00143] There were no differences in essential amino acids (EAA), branched chain amino acids (BCAA) or leucine plasma concentrations during the resting EB and ED trials. Plasma concentrations of EAA, BCAA and leucine increased above pre-exercise values between 20-120 minutes post exercise (see, FIGS. 3A-C) for both the 15 and 30 g treatments. The 30 g protein protocol resulted in higher aminoacidemia at 20 minutes post exercise (1.4 fold; P < 0.004) compared with 40 minutes post exercise following 15 g protein ingestion (1.7 fold; P < 0.001). Plasma concentration peaked 40-60 minutes post exercise (1.8-1.9 fold; P < 0.001) and remained above pre exercise values until 2 hours post exercise (1.6-1.9 fold; P < 0.02) in both the 15 g and 30 g treatments. Plasma EAA concentration increased in 30 g compared to 15 g between 20 minutes and 1 hour post exercise (1.2-1.3 fold; P < 0.03) (see, FIG. 3A). Plasma BCAA and leucine concentration followed a similar pattern but differences between 15 and 30 g remained until 2 hours post- exercise (1.2-1.8 fold P < 0.02) (see, FIGS. 3B and 3C). [00144] Muscle Myofibrillar Fractional Synthetic Rate.

[00145] Resting ED myofibrillar FSR was lower compared to resting EB (0.019 vs 0.026 %-h, 27%, P < 0.001) (see, FIG. 4). Resistance exercise in ED with placebo returned myofibrillar FSR to values comparable to resting EB in the acute post exercise recovery period. Resistance exercise followed by 15 g and 30 g protein ingestion increased post-exercise myofibrillar FSR -16% and -34% above resting EB, respectively (0.030 and 0.038 %-h respectively; P < 0.02). The 30 g protein treatment also increased myofibrillar FSR above 15 g by -14% (P < 0.003). There were no differences between genders in any of the treatments. Linear regression analysis showed a positive correlation between the quantity of protein ingested per kg of BM or FFM and myofibrillar FSR (r =0.43 and 0.42 respectively, P< 0.001) (see, FIGS. 5A-B).

[00146] Cell Signaling.

[00147] There were no differences in phosphorylation status between resting EB and ED for any of the proteins quantified. Akt ^Ser473 phosphorylation was higher than resting ED in all treatments 1 hour post exercise (1.8 -3.2 fold; P < 0.05 (see, FIG. 6A). Protein intake increased Akt phosphorylation above resting EB to a similar extent 1 hour post exercise regardless of protein quantity (15 g -2.1 fold, 30 g -2.4 fold; P < 0.02). There were similar effects on mTOR ^Ser244B and S6K ^{Thr 389} phosphorylation. Protein intake increased mTOR ^Ser244B phosphorylation above resting EB levels and placebo at the 1 hour post exercise time point (-2.5 fold from resting EB, -2 fold from PLl h; P < 0.006) (see, FIG. 6B). However, only the 30 g treatment prolonged the elevation in mTOR phosphorylation to 4 hours post-exercise (-2.1 fold; P < 0.05). The p70 S6K ^{Thr 389} phosphorylation increased above resting levels 1 hour and 4 hours following resistance exercise and protein ingestion (2.6-7 fold; P < 0.05) (see, FIG. 6C). Peak phosphorylation above rest was observed with 30 g protein at 1 hour post-exercise (7 fold; P < 0.001) and was higher than 15 g protein at the equivalent time point (1.8 fold, P=0.051). Phosphorylation of rpS6 ^{Ser 236/237} above resting EB was highest 1 hour after exercise with post exercise protein ingestion (12.5-19.2 fold; P < 0.001) (see, FIG. 6D). There were no differences in AMPK ¹ , 4EBPl ^ltirjb/4/ or eEF2 ^lfir5b phosphorylation at any time (data not shown).

[00148] mRNA Expression.

[00149] There were only minor changes in MuRF-1 mRNA content from resting EB but MuRF-1 was different from rest and select post exercise time points after 4 hours post-exercise recovery in the 15 g protein treatment (1.85 fold; P < 0.003) (see, FIG. 7A). Atrogin-1 mRNA content was higher than resting EB and ED, and when compared with 1 hour post exercise following 4 hours recovery (1.98-2.27 fold; P < 0.006) (see, FIG. 7B). There were no differences in system A amino acid transporter (SNAT2) mRNA content but in all treatments there was a decrease in system L amino acid transporter (LATl) mRNA content at 1 and 4 hours post-exercise compared to resting EB (-0.49-0.6; P < 0.03). In addition, LATl mRNA content following resistance exercise with PL (4 h) and 15 g protein ingestion (1 hour) was lower compared with resting ED (0.55-0.64; P < 0.04) (see, FIG. 7D).

[00150] In conclusion, Applicant has surprisingly found that during an energy deficit (ED) the loss of fat free mass (FFM) can be mitigated by exercise and consumption of higher dietary protein both of which are potent stimuli for MPS. The nature of the relationship between MPS, exercise, and protein during ED is unknown. Applicant has determined the effect of 5 days ED (30 kcal-kg ^"1 Fat Free Mass (FFM)-day energy availability (EA)) on basal MPS and cell signaling during ED and energy balance (EB; 45 kcal-kg ^"1 LBM- day EA) as well as after REX in the fasted state and with the ingestion of whey protein (15 and 30 g) in ED in young, healthy male (n=8) and female subjects (n=7). There were no sex-based differences (P = 0.3) for MPS. Basal rates of MPS were 27% lower in ED than EB (PO.001). REX in the fasted state restored rates of MPS to those measured in EB. However, ingestion of 15 and 30 g of protein after REX in ED increased MPS -16 and -34% above resting EB, (PO.02). Basal phosphorylation of mTOR- mediated signalling was similar in ED and EB. p70 S6K ^thr389 phosphorylation increased above EB with exercise and protein intake together (-2-7 fold; P<0.05) but not with REX alone. Applicant surprisingly concluded that short-term (e.g., 5 day) energy deficit down regulates basal MPS. However, a single bout of resistance exercise in ED rescues rates of MPS to values measured at rest in EB. The ingestion of protein after exercise further increases MPS above resting EB in a dose-dependent manner. The results of the present study highlight the effect of combining resistance exercise with increased post-exercise protein availability for enhancing rates of skeletal muscle protein synthesis during periods of energy restriction.

[00151] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.