The role of dietary plant and animal protein intakes on mitigating sarcopenia risk.

PURPOSE OF REVIEW: To highlight contemporary findings comparing the digestibility of animal and plant proteins, their stimulatory effects on muscle protein synthesis, and associations with sarcopenia. RECENT FINDINGS: Animal proteins are more digestible than plant proteins, resulting in greater amino acid availability and stimulation of muscle protein synthesis. However, isolated plant proteins, plant protein blends, and modified plant proteins enriched with indispensable amino acids can elicit comparable digestion and absorption kinetics to animal proteins. More research is needed to determine whether these modified plant protein sources can effectively mitigate sarcopenia risk. SUMMARY: Both animal and plant protein foods can be incorporated into a healthful eating plan that limits risk of age-related diseases, such as sarcopenia. Humans eat food rather than isolated nutrients; as such, considering the context of the overall diet and its impact on health, instead of solely focusing on individual nutrients in isolation, is important.

Physiological Limitations of Protein Foods Ounce Equivalents and the Underappreciated Role of Essential Amino Acid Density in Healthy Dietary Patterns.

Protein quality is an important component of protein intake to support growth, development, and maintenance of essential body tissues and functions. Therefore, protein quality should be emphasized as a key characteristic during protein food selection within the larger context of healthy dietary patterns, especially when considering the wide variance of protein quality across animal- and plant-based foods. However, the USDA Dietary Guidelines for Americans (DGA) do not address specific protein quality recommendations within their protein foods ounce equivalents guidance or as a component of Healthy U.S. Style, Healthy Vegetarian, and Healthy Mediterranean Style dietary patterns. In addition, the protein foods ounce equivalents within the DGA are not established on any obvious metabolic equivalency characteristic [i.e., energy, protein, or essential amino acid (EAA) content], which creates misleading messaging of equivalent functional and metabolic benefit across protein foods. EAA content is a key characteristic of protein quality and can be a practical focal point for protein intake recommendations and achieving healthy dietary patterns. This review discusses the importance of protein quality, the state of messaging within DGA recommendations, and proposes EAA density (i.e., EAA content relative to total energy) as one practical approach to improve current dietary recommendations. Two recent publications that evaluated the DGA protein foods ounce equivalents based on metabolic effect and their application within DGA recommended dietary patterns are discussed.

Essential amino acid-enriched whey enhances post-exercise whole-body protein balance during energy deficit more than iso-nitrogenous whey or a mixed-macronutrient meal: a randomized, crossover study.

BACKGROUND: The effects of ingesting varying essential amino acid (EAA)/protein-containing food formats on protein kinetics during energy deficit are undetermined. Therefore, recommendations for EAA/protein food formats necessary to optimize both whole-body protein balance and muscle protein synthesis (MPS) during energy deficit are unknown. We measured protein kinetics after consuming iso-nitrogenous amounts of free-form essential amino acid-enriched whey (EAA + W; 34.7 g protein, 24 g EAA sourced from whey and free-form EAA), whey (WHEY; 34.7 g protein, 18.7 g EAA), or a mixed-macronutrient meal (MEAL; 34.7 g protein, 11.4 g EAA) after exercise during short-term energy deficit. METHODS: Ten adults (mean ± SD; 21 ± 4 y; 25.7 ± 1.7 kg/m2) completed a randomized, double-blind crossover study consisting of three, 5 d energy-deficit periods (- 30 ± 3% of total energy requirements), separated by 14 d. Whole-body protein synthesis (PS), breakdown (PB), and net balance (NET) were determined at rest and in response to combination exercise consisting of load carriage treadmill walking, deadlifts, and box step-ups at the end of each energy deficit using L-[2H5]-phenylalanine and L-[2H2]-tyrosine infusions. Treatments were ingested immediately post-exercise. Mixed-muscle protein synthesis (mixed-MPS) was measured during exercise through recovery. RESULTS: Change (Δ postabsorptive + exercise to postprandial + recovery [mean treatment difference (95%CI)]) in whole-body (g/180 min) PS was 15.8 (9.8, 21.9; P = 0.001) and 19.4 (14.8, 24.0; P = 0.001) greater for EAA + W than WHEY and MEAL, respectively, with no difference between WHEY and MEAL. ΔPB was - 6.3 (- 11.5, - 1.18; P = 0.02) greater for EAA + W than WHEY and - 7.7 (- 11.9, - 3.6; P = 0.002) greater for MEAL than WHEY, with no difference between EAA + W and MEAL. ΔNET was 22.1 (20.5, 23.8; P = 0.001) and 18.0 (16.5, 19.5; P = 0.00) greater for EAA + W than WHEY and MEAL, respectively, while ΔNET was 4.2 (2.7, 5.6; P = 0.001) greater for MEAL than WHEY. Mixed-MPS did not differ between treatments. CONCLUSIONS: While mixed-MPS was similar across treatments, combining free-form EAA with whey promotes greater whole-body net protein balance during energy deficit compared to iso-nitrogenous amounts of whey or a mixed-macronutrient meal. TRIAL REGISTRATION: ClinicalTrials.gov, Identifier no. NCT04004715 . Retrospectively registered 28 June 2019, first enrollment 6 June 2019.

Effects of high versus standard essential amino acid intakes on whole-body protein turnover and mixed muscle protein synthesis during energy deficit: A randomized, crossover study.

BACKGROUND & AIMS: Consuming 0.10-0.14 g essential amino acids (EAA)/kg/dose (0.25-0.30 g protein/kg/dose) maximally stimulates muscle protein synthesis (MPS) during energy balance. Whether consuming EAA beyond that amount enhances MPS and whole-body anabolism following energy deficit is unknown. The aims of this study were to determine the effects of standard and high EAA ingestion on mixed MPS and whole-body protein turnover following energy deficit. DESIGN: Nineteen males (mean ± SD; 23 ± 5 y; 25.4 ± 2.7 kg/m2) completed a randomized, double-blind crossover study consisting of two, 5-d energy deficits (-30 ± 4% of total energy requirements), separated by 14-d. Following each energy deficit, mixed MPS and whole-body protein synthesis (PS), breakdown (PB), and net balance (NET) were determined at rest and post-resistance exercise (RE) using primed, constant L-[2H5]-phenylalanine and L-[2H2]-tyrosine infusions. Beverages providing standard (0.1 g/kg, 7.87 ± 0.87 g) or high (0.3 g/kg, 23.5 ± 2.54 g) EAA were consumed post-RE. Circulating EAA were measured. RESULTS: Postabsorptive mixed MPS (%/h) at rest was not different (P = 0.67) between treatments. Independent of EAA, postprandial mixed MPS at rest (standard EAA, 0.055 ± 0.01; high EAA, 0.061 ± 0.02) and post-RE (standard EAA, 0.055 ± 0.01; high EAA, 0.065 ± 0.02) were greater than postabsorptive mixed MPS at rest (P = 0.02 and P = 0.01, respectively). Change in (Δ postabsorptive) whole-body (g/180 min) PS and PB was greater for high than standard EAA [mean treatment difference (95% CI), 3.4 (2.3, 4.4); P = 0.001 and -15.6 (-17.8, -13.5); P = 0.001, respectively]. NET was more positive for high than standard EAA [19.0 (17.3, 20.7); P = 0.001]. EAA concentrations were greater in high than standard EAA (P = 0.001). CONCLUSIONS: These data demonstrate that high compared to standard EAA ingestion enhances whole-body protein status during underfeeding. However, the effects of consuming high and standard EAA on mixed MPS are the same during energy deficit. CLINICAL TRIAL REGISTRY: NCT03372928, https://clinicaltrials.gov.

Dietary Protein and Muscle Mass: Translating Science to Application and Health Benefit.

Adequate consumption of dietary protein is critical for the maintenance of optimal health during normal growth and aging. The current Recommended Dietary Allowance (RDA) for protein is defined as the minimum amount required to prevent lean body mass loss, but is often misrepresented and misinterpreted as a recommended optimal intake. Over the past two decades, the potential muscle-related benefits achieved by consuming higher-protein diets have become increasingly clear. Despite greater awareness of how higher-protein diets might be advantageous for muscle mass, actual dietary patterns, particularly as they pertain to protein, have remained relatively unchanged in American adults. This lack of change may, in part, result from confusion over the purported detrimental effects of higher-protein diets. This manuscript will highlight common perceptions and benefits of dietary protein on muscle mass, address misperceptions related to higher-protein diets, and comment on the translation of academic advances to real-life application and health benefit. Given the vast research evidence supporting the positive effects of dietary protein intake on optimal health, we encourage critical evaluation of current protein intake recommendations and responsible representation and application of the RDA as a minimum protein requirement rather than one determined to optimally meet the needs of the population.

Recent Advances in the Characterization of Skeletal Muscle and Whole-Body Protein Responses to Dietary Protein and Exercise during Negative Energy Balance.

In a review published in 2012, we concluded that higher-protein diets preserve muscle mass during energy deficit via stimulated mammalian target of rapamycin complex 1 signaling, coincident increased muscle protein synthesis (PS), inhibited ubiquitin-mediated proteolysis, and suppressed muscle protein breakdown (PB). Since then, there have been significant advances in understanding the fundamental effects of higher-protein diets, with or without exercise training, on muscle and whole-body protein homeostasis during negative energy balance. Therefore, an update on the evolution of this field of research is warranted to better inform recommendations on best practices for healthy weight loss and muscle preservation. We will review the most recent studies examining the effects of higher-protein diets and negative energy balance on body composition, muscle PS, muscle PB, associated intracellular regulatory pathway activities, and whole-body protein homeostasis. In addition to critically analyzing contemporary findings, knowledge gaps and opportunities for continued research will be identified. Overall, the newest research confirms that consuming higher-protein diets, particularly when coupled with resistance exercise, preserves muscle mass and maintains whole-body protein homeostasis during moderate energy deficits (i.e., normal weight loss). However, these newer findings also indicate that as the magnitude of energy deficit increases, the efficacy of higher-protein diets for mitigating losses of fat-free mass is diminished. Further, recent results suggest that alterations in muscle PS, more so than muscle PB, may be primarily responsible for changes in muscle mass that occur in response to negative energy balance.

PI3K-AKT-FOXO1 pathway targeted by skeletal muscle microRNA to suppress proteolytic gene expression in response to carbohydrate intake during aerobic exercise.

Ingesting protein and carbohydrate together during aerobic exercise suppresses the expression of specific skeletal muscle microRNA and promotes muscle hypertrophy. Determining whether there are independent effects of carbohydrate and protein on microRNA will allow for a clearer understanding of the mechanistic role microRNA serve in regulating skeletal muscle protein synthetic and proteolytic responses to nutrition and exercise. This study determined skeletal muscle microRNA responses to aerobic exercise with or without carbohydrate, and recovery whey protein (WP). Seventeen males were randomized to consume carbohydrate (CHO; 145 g; n = 9) or non-nutritive control (CON; n = 8) beverages during exercise. Muscle was collected before (BASE) and after 80 min of steady-state exercise (1.7 ± 0.3 V̇O2  L·min-1 ) followed by a 2-mile time trial (17.9 ± 3.5 min; POST), and 3-h into recovery after consuming WP (25 g; REC). RT-qPCR was used to determine microRNA and mRNA expression. Bioinformatics analysis was conducted using the mirPath software. Western blotting was used to assess protein signaling. The expression of six microRNA (miR-19b-3p, miR-99a-5p, miR-100-5p, miR-222-3p, miR-324-3p, and miR-486-5p) were higher (P < 0.05) in CHO compared to CON, all of which target the PI3K-AKT, ubiquitin proteasome, FOXO, and mTORC1 pathways. p-AKTThr473 and p-FOXO1Thr24 were higher (P < 0.05) in POST CHO compared to CON. The expression of PTEN was lower (P < 0.05) in REC CHO than CON, while MURF1 was lower (P < 0.05) POST CHO than CON. These findings suggest the mechanism by which microRNA facilitate skeletal muscle adaptations in response to exercise with carbohydrate and protein feeding is by inhibiting markers of proteolysis.

Altitude Acclimatization Alleviates the Hypoxia-Induced Suppression of Exogenous Glucose Oxidation During Steady-State Aerobic Exercise.

This study investigated how high-altitude (HA, 4300 m) acclimatization affected exogenous glucose oxidation during aerobic exercise. Sea-level (SL) residents (n = 14 men) performed 80-min, metabolically matched exercise ( V˙ O2 ∼ 1.7 L/min) at SL and at HA < 5 h after arrival (acute HA, AHA) and following 22-d of HA acclimatization (chronic HA, CHA). During HA acclimatization, participants sustained a controlled negative energy balance (-40%) to simulate the "real world" conditions that lowlanders typically experience during HA sojourns. During exercise, participants consumed carbohydrate (CHO, n = 8, 65.25 g fructose + 79.75 g glucose, 1.8 g carbohydrate/min) or placebo (PLA, n = 6). Total carbohydrate oxidation was determined by indirect calorimetry and exogenous glucose oxidation by tracer technique with 13C. Participants lost (P ≤ 0.05, mean ± SD) 7.9 ± 1.9 kg body mass during the HA acclimatization and energy deficit period. In CHO, total exogenous glucose oxidized during the final 40 min of exercise was lower (P < 0.01) at AHA (7.4 ± 3.7 g) than SL (15.3 ± 2.2 g) and CHA (12.4 ± 2.3 g), but there were no differences between SL and CHA. Blood glucose and insulin increased (P ≤ 0.05) during the first 20 min of exercise in CHO, but not PLA. In CHO, glucose declined to pre-exercise concentrations as exercise continued at SL, but remained elevated (P ≤ 0.05) throughout exercise at AHA and CHA. Insulin increased during exercise in CHO, but the increase was greater (P ≤ 0.05) at AHA than at SL and CHA, which did not differ. Thus, while acute hypoxia suppressed exogenous glucose oxidation during steady-state aerobic exercise, that hypoxic suppression is alleviated following altitude acclimatization and concomitant negative energy balance.

Muscle Fn14 gene expression is associated with fat-free mass retention during energy deficit at high altitude.

Intramuscular factors that modulate fat-free mass (FFM) loss in lowlanders exposed to energy deficit during high-altitude (HA) sojourns remain unclear. Muscle inflammation may contribute to FFM loss at HA by inducing atrophy and inhibiting myogenesis via the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible protein 14 (Fn14). To explore whether muscle inflammation modulates FFM loss reportedly developing during HA sojourns, muscle inflammation, myogenesis, and proteolysis were assessed in 16 men at sea level (SL) and following 21 days of energy deficit (-1862 ± 525 kcal/days) at high altitude (HA, 4300 m). Total body mass (TBM), FFM, and fat mass (FM) were assessed using DEXA. Gene expression and proteolytic enzymatic activities were assessed in muscle samples collected at rest at SL and HA. Participants lost 7.2 ± 1.8 kg TBM (P < 0.05); 43 ± 30% and 57 ± 30% of the TBM lost was FFM and FM, respectively. Fn14, TWEAK, TNF alpha-receptor (TNFα-R), TNFα, MYOGENIN, and paired box protein-7 (PAX7) were upregulated (P < 0.05) at HA compared to SL. Stepwise linear regression identified that Fn14 explained the highest percentage of variance in FFM loss (r2  = 0.511, P < 0.05). Dichotomization of volunteers into HIGH and LOW Fn14 gene expression indicated HIGH lost less FFM and more FM (28 ± 28% and 72 ± 28%, respectively) as a proportion of TBM loss than LOW (58 ± 26% and 42 ± 26%; P < 0.05) at HA. MYOGENIN gene expression was also greater for HIGH versus LOW (P < 0.05). These data suggest that heightened Fn14 gene expression is not catabolic and may protect FFM during HA sojourns.

Severe energy deficit at high altitude inhibits skeletal muscle mTORC1-mediated anabolic signaling without increased ubiquitin proteasome activity.

Muscle loss at high altitude (HA) is attributable to energy deficit and a potential dysregulation of anabolic signaling. Exercise and protein ingestion can attenuate the effects of energy deficit on muscle at sea level (SL). Whether these effects are observed when energy deficit occurs at HA is unknown. To address this, muscle obtained from lowlanders ( n = 8 males) at SL, acute HA (3 h, 4300 m), and chronic HA (21 d, -1766 kcal/d energy balance) before [baseline (Base)] and after 80 min of aerobic exercise followed by a 2-mile time trial [postexercise (Post)] and 3 h into recovery (Rec) after ingesting whey protein (25 g) were analyzed using standard molecular techniques. At SL, Post, and REC, p-mechanistic target of rapamycin (mTOR)Ser2448, p-p70 ribosomal protein S6 kinase (p70S6K)Ser424/421, and p-ribosomal protein S6 (rpS6)Ser235/236 were similar and higher ( P < 0.05) than Base. At acute HA, Post p-mTORSer2448 and Post and REC p-p70S6KSer424/421 were not different from Base and lower than SL ( P < 0.05). At chronic HA, Post and Rec p-mTORSer2448 and p-p70S6KSer424/421 were not different from Base and lower than SL, and, independent of time, p-rpS6Ser235/236 was lower than SL ( P < 0.05). Post proteasome activity was lower ( P < 0.05) than Base and Rec, independent of phase. Our findings suggest that HA exposure induces muscle anabolic resistance that is exacerbated by energy deficit during acclimatization, with no change in proteolysis.-Margolis, L. M., Carbone, J. W., Berryman, C. E., Carrigan, C. T., Murphy, N. E., Ferrando, A. A., Young, A. J., Pasiakos, S. M. Severe energy deficit at high altitude inhibits skeletal muscle mTORC1-mediated anabolic signaling without increased ubiquitin proteasome activity.

Appetite Suppression and Altered Food Preferences Coincide with Changes in Appetite-Mediating Hormones During Energy Deficit at High Altitude, But Are Not Affected by Protein Intake.

Karl, J. Philip, Renee E. Cole, Claire E. Berryman, Graham Finlayson, Patrick N. Radcliffe, Matthew T. Kominsky, Nancy E. Murphy, John W. Carbone, Jennifer C. Rood, Andrew J. Young, and Stefan M. Pasiakos. Appetite suppression and altered food preferences coincide with changes in appetite-mediating hormones during energy deficit at high altitude, but are not affected by protein intake. High Alt Med Biol. 19:156-169, 2018.-Anorexia and unintentional body weight loss are common during high altitude (HA) sojourn, but underlying mechanisms are not fully characterized, and the impact of dietary macronutrient composition on appetite regulation at HA is unknown. This study aimed to determine the effects of a hypocaloric higher protein diet on perceived appetite and food preferences during HA sojourn and to examine longitudinal changes in perceived appetite, appetite mediating hormones, and food preferences during acclimatization and weight loss at HA. Following a 21-day level (SL) period, 17 unacclimatized males ascended to and resided at HA (4300 m) for 22 days. At HA, participants were randomized to consume measured standard-protein (1.0 g protein/kg/d) or higher protein (2.0 g/kg/d) hypocaloric diets (45% carbohydrate, 30% energy restriction) and engaged in prescribed physical activity to induce an estimated 40% energy deficit. Appetite, food preferences, and appetite-mediating hormones were measured at SL and at the beginning and end of HA. Diet composition had no effect on any outcome. Relative to SL, appetite was lower during acute HA (days 0 and 1), but not different after acclimatization and weight loss (HA day 18), and food preferences indicated an increased preference for sweet- and low-protein foods during acute HA, but for high-fat foods after acclimatization and weight loss. Insulin, leptin, and cholecystokinin concentrations were elevated during acute HA, but not after acclimatization and weight loss, whereas acylated ghrelin concentrations were suppressed throughout HA. Findings suggest that appetite suppression and altered food preferences coincide with changes in appetite-mediating hormones during energy deficit at HA. Although dietary protein intake did not impact appetite, the possible incongruence with food preferences at HA warrants consideration when developing nutritional strategies for HA sojourn.

Severe negative energy balance during 21 d at high altitude decreases fat-free mass regardless of dietary protein intake: a randomized controlled trial.

In this 2-phase randomized controlled study, we examined whether consuming a higher-protein (HP) diet would attenuate fat-free mass (FFM) loss during energy deficit (ED) at high altitude (HA) in 17 healthy males (mean ± sd: 23 ± 6 yr; 82 ± 14 kg). During phase 1 at sea level (SL, 55 m), participants consumed a eucaloric diet providing standard protein (SP; 1.0 g protein/kg,) for 21 d. During phase 2, participants resided at HA (4300 m) for 22 d and were randomly assigned to either an SP or HP (2.0 g protein/kg) diet designed to elicit a 40% ED. Body composition, substrate oxidation, and postabsorptive whole-body protein kinetics were measured. Participants were weight stable during SL and lost 7.9 ± 1.9 kg ( P < 0.01) during HA, regardless of dietary protein intake. Decrements in whole-body FFM (3.6 ± 2.4 kg) and fat mass (3.6 ± 1.3 kg) were not different between SP and HP. HP oxidized 0.95 ± 0.32 g protein/kg per day more than SP and whole-body net protein balance was more negative for HP than for SP ( P < 0.01). Based on changes in body energy stores, the overall ED was 70% (-1849 ± 511 kcal/d, no group differences). Consuming an HP diet did not protect FFM during severe ED at HA.-Berryman, C. E., Young, A. J., Karl, J. P., Kenefick, R. W., Margolis, L. M., Cole, R. E., Carbone, J. W., Lieberman, H. R., Kim, I.-Y., Ferrando, A. A., Pasiakos, S. M. Severe negative energy balance during 21 d at high altitude decreases fat-free mass regardless of dietary protein intake: a randomized controlled trial.

Muscle Protein Turnover and the Molecular Regulation of Muscle Mass during Hypoxia.

: Effects of environmental hypoxia on fat-free mass are well studied. Negative energy balance, increased nitrogen excretion, and fat-free mass loss are commonly observed in lowlanders sojourning at high altitude. Reductions in fat-free mass can be minimized if energy consumption matches energy expenditure. However, in nonresearch settings, achieving energy balance during high-altitude sojourns is unlikely, and myofibrillar protein mass is usually lost, but the mechanisms accounting for the loss of muscle mass are not clear. At sea level, negative energy balance reduces basal and blunts postprandial muscle protein synthesis, with no relevant change in muscle protein breakdown. Downregulations in muscle protein synthesis and loss of fat-free mass during energy deficit at sea level are largely overcome by consuming at least twice the recommended dietary allowance for protein. Hypoxia may increase or not affect resting muscle protein synthesis, blunt postexercise muscle protein synthesis, and markedly increase proteolysis independent of energy status. Hypoxia-induced mTORC1 dysregulation and an upregulation in calpain- and ubiquitin proteasome-mediated proteolysis may drive catabolism in lowlanders sojourning at high altitude. However, the combined effects of energy deficit, exercise, and dietary protein manipulations on the regulation of muscle protein turnover have never been studied at high altitude. This article reviews the available literature related to the effects of high altitude on fat-free mass, highlighting contemporary studies that assessed the influence of altitude exposure (or hypoxia) on muscle protein turnover and intramuscular regulation of muscle mass. Knowledge gaps are addressed, and studies to identify effective and feasible countermeasures to hypoxia-induced muscle loss are discussed.

Paleolithic nutrition improves plasma lipid concentrations of hypercholesterolemic adults to a greater extent than traditional heart-healthy dietary recommendations.

Recent research suggests that traditional grain-based heart-healthy diet recommendations, which replace dietary saturated fat with carbohydrate and reduce total fat intake, may result in unfavorable plasma lipid ratios, with reduced high-density lipoprotein (HDL) and an elevation of low-density lipoprotein (LDL) and triacylglycerols (TG). The current study tested the hypothesis that a grain-free Paleolithic diet would induce weight loss and improve plasma total cholesterol, HDL, LDL, and TG concentrations in nondiabetic adults with hyperlipidemia to a greater extent than a grain-based heart-healthy diet, based on the recommendations of the American Heart Association. Twenty volunteers (10 male and 10 female) aged 40 to 62 years were selected based on diagnosis of hypercholesterolemia. Volunteers were not taking any cholesterol-lowering medications and adhered to a traditional heart-healthy diet for 4 months, followed by a Paleolithic diet for 4 months. Regression analysis was used to determine whether change in body weight contributed to observed changes in plasma lipid concentrations. Differences in dietary intakes and plasma lipid measures were assessed using repeated-measures analysis of variance. Four months of Paleolithic nutrition significantly lowered (P < .001) mean total cholesterol, LDL, and TG and increased (P < .001) HDL, independent of changes in body weight, relative to both baseline and the traditional heart-healthy diet. Paleolithic nutrition offers promising potential for nutritional management of hyperlipidemia in adults whose lipid profiles have not improved after following more traditional heart-healthy dietary recommendations.

Assessment of skeletal muscle proteolysis and the regulatory response to nutrition and exercise.

Skeletal muscle proteolysis is highly regulated, involving complex intramuscular proteolytic systems that recognize and degrade muscle proteins, and recycle free amino acid precursors for protein synthesis and energy production. Autophagy-lysosomal, calpain, and caspase systems are contributors to muscle proteolysis, although the ubiquitin proteasome system (UPS) is the primary mechanism by which actomyosin fragments are degraded in healthy muscle. The UPS is sensitive to mechanical force and nutritional deprivation, as recent reports have demonstrated increased proteolytic gene expression and activity of the UPS in response to resistance and endurance exercise, and short-term negative energy balance. However, consuming dietary protein alone (or free amino acids), or as a primary component of a mixed meal, may attenuate intramuscular protein loss by down-regulating proteolytic gene expression and the catabolic activity of the UPS. Although these studies provide novel insight regarding the intramuscular regulation of skeletal muscle mass, the role of proteolysis in the regulation of skeletal muscle protein turnover in healthy human muscle is not well described. This article provides a contemporary review of the intramuscular regulation of skeletal muscle proteolysis in healthy muscle, methodological approaches to assess proteolysis, and highlights the effects of nutrition and exercise on skeletal muscle proteolysis.

Effects of short-term energy deficit on muscle protein breakdown and intramuscular proteolysis in normal-weight young adults.

The effects of short-term energy deficit (ED) on direct measures of muscle proteolysis and the intracellular mechanisms by which muscle proteins are degraded at rest and following aerobic exercise are not well described. This study evaluated the effects of a short-term diet-induced ED, on muscle fractional breakdown rate (FBR), intramuscular 26S proteasome activity, caspase-3 activation, and PSMA2 and MAFbx expression at rest, in the postabsorptive state, and following a single bout of moderate aerobic exercise (45 min at 65% peak oxygen uptake). Six men and 4 women participated in two 10-day diet interventions: weight maintenance (WM) followed by ED (80% estimated energy requirements). Dietary protein (1.5 g·kg(-1)·day(-1)) intake was constant for WM and ED. Mixed muscle FBR, proteasome activity, and intracellular proteolytic factor expression were measured using stable isotope methodology, fluorescent enzyme activity assays, and Western blotting, respectively. Overall, FBR and caspase-3 activation increased 60% and 11%, respectively, in response to ED (P < 0.05), but were not influenced by exercise. During ED, 26S proteasome α-subunit PSMA2 expression was 25% higher (P < 0.05) after exercise compared with rest. Exercise did not influence PSMA2 expression during WM, and MAFbx expression and 26S proteasome activity were not affected by ED or exercise. These data illustrate the effects of short-term, moderate ED on muscle protein degradation. In the context of skeletal muscle integrity during weight loss interventions, this work demonstrates a need for further investigations aimed at mitigating muscle loss associated with energy deficit imposed for intentional reduction of total body weight.

Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis.

This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×-RDA), or 2.4 (3×-RDA) g · kg(-1) · d(-1) for 31 d. A 10-d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)-mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence-based assays, immunoblot analysis, and real-time qRT-PCR. In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up-regulation of UPS-associated gene expression, as mRNA expression of the atrogenes muscle RING finger-1 (MuRF1) and atrogin-1 were 1.2- and 1.3-fold higher (P<0.05) for ED than for WM. However, mixed-meal consumption attenuated UPS-mediated proteolysis, independent of energy status or dietary protein, as the activities of the 26S proteasome subunits ß1, ß2, and ß5 were lower (P<0.05) for fed than for fasted. Muscle protein ubiquitylation was also 45% lower (P<0.05) for fed than for fasted, regardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin-1 mRNA expression during ED, consuming a protein-containing mixed meal attenuates Ub-mediated proteolysis.

Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial.

The purpose of this work was to determine the effects of varying levels of dietary protein on body composition and muscle protein synthesis during energy deficit (ED). A randomized controlled trial of 39 adults assigned the subjects diets providing protein at 0.8 (recommended dietary allowance; RDA), 1.6 (2×-RDA), and 2.4 (3×-RDA) g kg(-1) d(-1) for 31 d. A 10-d weight-maintenance (WM) period was followed by a 21 d, 40% ED. Body composition and postabsorptive and postprandial muscle protein synthesis were assessed during WM (d 9-10) and ED (d 30-31). Volunteers lost (P<0.05) 3.2 ± 0.2 kg body weight during ED regardless of dietary protein. The proportion of weight loss due to reductions in fat-free mass was lower (P<0.05) and the loss of fat mass was higher (P<0.05) in those receiving 2×-RDA and 3×-RDA compared to RDA. The anabolic muscle response to a protein-rich meal during ED was not different (P>0.05) from WM for 2×-RDA and 3×-RDA, but was lower during ED than WM for those consuming RDA levels of protein (energy × protein interaction, P<0.05). To assess muscle protein metabolic responses to varied protein intakes during ED, RDA served as the study control. In summary, we determined that consuming dietary protein at levels exceeding the RDA may protect fat-free mass during short-term weight loss.

Skeletal muscle responses to negative energy balance: effects of dietary protein.

Sustained periods of negative energy balance decrease body mass due to losses of both fat and skeletal muscle mass. Decreases in skeletal muscle mass are associated with a myriad of negative consequences, including suppressed basal metabolic rate, decreased protein turnover, decreased physical performance, and increased risk of injury. Decreases in skeletal muscle mass in response to negative energy balance are due to imbalanced rates of muscle protein synthesis and degradation. However, the underlying physiological mechanisms contributing to the loss of skeletal muscle during energy deprivation are not well described. Recent studies have demonstrated that consuming dietary protein at levels above the current recommended dietary allowance (0.8 g · kg(-1) · d(-1)) may attenuate the loss of skeletal muscle mass by affecting the intracellular regulation of muscle anabolism and proteolysis. However, the specific mechanism by which increased dietary protein spares skeletal muscle through enhanced molecular control of muscle protein metabolism has not been elucidated. This article reviews the available literature related to the effects of negative energy balance on skeletal muscle mass, highlighting investigations that assessed the influence of varying levels of dietary protein on skeletal muscle protein metabolism. Further, the molecular mechanisms that may contribute to the regulation of skeletal muscle mass in response to negative energy balance and alterations in dietary protein level are described.

Chocolate milk and endurance exercise recovery: protein balance, glycogen, and performance.

PURPOSE: This study examined effects of fat-free chocolate milk (MILK) consumption on kinetic and cellular markers of protein turnover, muscle glycogen, and performance during recovery from endurance exercise. METHODS: Male runners participated in two trials separated by 1 wk and consumed either MILK or a nonnitrogenous isocaloric carbohydrate (CHO) control beverage (CON) after a 45-min run at 65% of V˙O(2peak). Postexercise muscle protein fractional synthetic rate (FSR) and whole-body protein turnover were determined during 3 h of recovery using muscle biopsies and primed constant infusions of L-[ring-²H5]phenylalanine and L-[1-¹³C]leucine, respectively. Phosphorylation of translational signaling proteins and activity of proteolytic molecules were determined using Western blotting and enzymatic activity assays. Muscle glycogen was quantified, and treadmill time to exhaustion was determined after the recovery period. RESULTS: Consuming MILK after exercise resulted in higher mixed muscle FSR with lower whole-body proteolysis and synthesis compared with CON (P ≤ 0.05). Phosphorylation of eIF4E-BP1 and FOXO3a was higher for MILK (P < 0.01), whereas Akt phosphorylation was lower during recovery regardless of dietary treatment (P < 0.05). Enzymatic activity assays indicated lower caspase-3 activity during recovery for MILK (P < 0.01) and higher 26S proteasome activity for CON (P < 0.01). Muscle glycogen was not affected by either dietary treatment; however, time to exhaustion was greater for MILK than for CON (P < 0.05). CONCLUSIONS: The effects of consumption of MILK after endurance exercise on FSR, signaling molecules of skeletal muscle protein turnover, leucine kinetics, and performance measures suggest unique benefits of milk compared with a CHO-only beverage.