J Clin Endocrin Metab. First published ahead of print October 9, 2013 as doi: /jc

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1 J Clin Endocrin Metab. First published ahead of print October 9, 2013 as doi: /jc Disuse impairs the muscle protein synthetic response to protein ingestion in healthy men Benjamin T Wall 1, Tim Snijders 1, Joan MG Senden 1, Chris LP Ottenbros 2, Annemie P Gijsen 3, Lex B Verdijk 1, and Luc JC van Loon 1 1Department of Human Movement Sciences, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Maastricht, 6200 MD, The Netherlands.; 2 Department of Surgery, Maastricht University Medical Centre, Maastricht, 6200 MD, The Netherlands.; 3 Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Maastricht, 6200 MD, The Netherlands. Background: Disuse leads to rapid skeletal muscle atrophy which brings about numerous negative health consequences. Muscle disuse atrophy is, at least in part, attributed to a decline in basal (post-absorptive) muscle protein synthesis rates. However, it remains to be determined whether muscle disuse also impairs the muscle protein synthetic response to dietary protein ingestion. Purpose: We assessed muscle protein synthesis rates following protein ingestion before and after a period of disuse in humans. Methods: Twelve healthy, young (24 1 y) men underwent a 14 day period of one-legged knee immobilization by way of a full leg cast. Before and after the immobilization period, quadriceps cross sectional area (CSA), muscle strength, skeletal muscle protein synthesis rates, and associated intramuscular molecular signaling were assessed. Continuous infusions of L-[ring- 2 H 5 ]phenylalanine were applied to assess mixed muscle protein fractional synthetic rates (FSRs) following the ingestion of 20 g dietary protein. Results: Immobilization led to an (P 0.001) and % (P 0.001) decrease in quadriceps muscle CSA and strength, respectively. Immobilization resulted in a 31 12% reduction in post-prandial muscle protein synthesis rates (from to %. h -1 ; P 0.05). These findings were observed without any discernible changes in the skeletal muscle phosphorylation status of mammalian target of rapamycin (mtor) or p70 ribosomal protein S6 kinase (p70 S6K ). Conclusions: A short period of muscle disuse impairs the muscle protein synthetic response to dietary protein intake in vivo in healthy young men. Thus, anabolic resistance to protein ingestion contributes significantly to the loss of muscle mass that is observed during disuse. Several situations, such as the recovery from injury and illness, necessitate periods of muscle disuse or unloading in (otherwise) healthy individuals. Under such conditions, rapid skeletal muscle atrophy (1, 2), reduced functional strength (1, 2), the onset of insulin resistance (3), a decline in basal metabolic rate (4) and an increase in body fat mass (5) have been reported. For these reasons, muscle disuse forms a significant health concern in several clinically compromised populations, with the elderly of particular relevance. Indeed, in elderly and/or patient populations who are already challenged by carrying out their daily living activities, brief periods of limb immobilization ISSN Print X ISSN Online Printed in U.S.A. Copyright 2013 by The Endocrine Society Received May 2, Accepted September 25, or bed-rest can lead to the loss of independence and a decline in overall health status (6). The prevalence of various successive periods of bed rest or immobilization has been suggested to contribute significantly to the development of age-related sarcopenia and the ensuing negative impact on functional capacity and quality of life (QOL) (6, 7). Therefore, it is of importance to elucidate the physiological mechanisms responsible for muscle disuse atrophy. Mechanistically, any substantial loss of skeletal muscle mass must be underpinned by a chronic imbalance between muscle protein synthesis and breakdown rates. A decline in basal muscle protein synthesis rates has been Abbreviations: FSR, fractional synthetic rate doi: /jc J Clin Endocrinol Metab jcem.endojournals.org 1 Copyright (C) 2013 by The Endocrine Society

2 2 disuse and post-prandial muscle protein accretion J Clin Endocrinol Metab reported following days of bed-rest (5, 8 11) or lower limb immobilization (2, 12 14) without any apparent changes in muscle protein breakdown (5, 11). Consequently, it is generally believed that muscle atrophy during a period of disuse is primarily due to impairments in the regulation of skeletal muscle protein synthesis (15, 16). To date, measurements of muscle protein turnover during disuse have primarily been determined in the basal, postabsorptive state. Food intake, and dietary protein in particular, stimulates muscle protein synthesis rates thereby allowing net muscle protein accretion (17 20). In this way, the muscle protein synthetic response to meal ingestion is recognized as a key factor allowing normal maintenance of muscle mass. Recent work suggests that the muscle protein synthetic response to amino acid administration becomes blunted following a period of disuse (13, 21). As a consequence, one possible explanation for the loss of muscle mass during a period of disuse could be a reduced sensitivity of the skeletal muscle protein synthetic machinery to food intake. We hypothesized that two weeks of muscle disuse would impair the muscle protein synthetic response to the ingestion of a single meal-like bolus of dietary protein in healthy men. In the present study we assessed postprandial muscle protein synthesis rates in twelve healthy young males following the ingestion of 20 g of casein protein before and after a two week period of limb immobilization. Continuous infusions with L-[ring- 2 H 5 ]phenylalanine were combined with muscle and blood sampling to assess mixed muscle protein fractional synthetic rates (FSRs) following the ingestion of 20 g casein protein before and after a 2 week period of one-legged knee immobilization. This is the first study to show that muscle disuse leads to impairments in the muscle protein synthetic response to the ingestion of a single meal like amount of dietary protein. Materials and Methods Subjects Twelve healthy, young (24 1 year), recreationally active men volunteered to participate in the present study. Subjects characteristics are presented in Table 2 (supplementary information). All subjects were fully informed of the nature and possible risks of the experimental procedures, before providing written informed consent. Subjects were screened to exclude any person with lower limb and/or back injuries sustained within a year prior to beginning the study, a (family) history of thrombosis/cardiovascular disease, use of anticoagulants, musculoskeletal/orthopaedic/ hemostatic disorders, or participation in any regular resistance training program within six months of beginning the study. The study was approved by the Medical Ethics Committee of the Maastricht University Medical Centre, Maastricht, the Netherlands. Experimental design Subjects participated in an experiment which comprised two experimental visits separated by a two week period of one legged knee immobilization by means of a full leg cast. During these experimental visits, infusions of L-[ring- 2 H 5 ]phenylalanine were applied to assess mixed muscle protein fractional synthetic rates (FSRs) following the ingestion of 20 g casein protein. In addition, a series of measurements were carried out to determine changes in muscle mass and strength during the immobilization period. Muscle mass and strength Approximately ten days prior to the immobilization period, subjects participated in two sessions, separated by 2 days, to assess muscle mass and strength. During the first visit, subjects arrived at the laboratory at hours in the fasted state and body weight was measured, a single slice CT-scan (Philips Brilliance 64, Philips Medical Systems, Best, the Netherlands) was performed to assess upper leg muscle cross-sectional area (CSA), and body composition was determined by DXA scan (Hologic Inc., Bedford, USA) as previously described (22). All subjects were then instructed and familiarized with safe lifting technique for the leg extension exercise (Technogym, Rotterdam, the Netherlands). Maximum strength was estimated using the multiple repetitions testing procedure (23) for each leg separately and confirmed during the second visit two days later (24). Finally, the leg to be immobilized was selected at random (counterbalanced for left and right) and subjects were instructed and familiarized with the use of crutches. Two days after cast removal, measures of muscle mass and strength were repeated in the same manner. (For complete details of muscle mass and strength measurements please see supplementary information). Habitual dietary intake Daily energy intake and macronutrient composition of the diet were calculated based upon dietary intake questionnaires filled out under the supervision of a dietician for two days prior to and during the last two days of the immobilization period. These records were subsequently analyzed using DieetInzicht Software, based on NEVO Table Experimental visits All subjects received the same standardized meal the evening prior to the stable isotope infusion experimental visits (33 2 kj. kg -1 body weight, providing 44 energy% (En%) carbohydrate, 22 En% protein, and 34 En% fat) and were instructed to refrain from strenuous physical activity and avoid alcohol intake for two days prior to the experimental visits. Two days prior to the immobilization period, and at least 3 days following the assessment of leg muscle strength, subjects arrived at the laboratory by car at 08:00 hours for the first of two identical stable isotope infusion experimental visits. A polytetrafluoroethylene catheter was inserted into an antecubital vein for stable-isotope infusion. A second catheter was inserted into a heated dorsal hand vein of the contralateral arm after which the hand was placed in a hot box (60 C) for arterialized blood sampling (25). After basal blood samples were collected (t 120 minutes), the plasma phenylalanine pool was primed with a single intravenous (IV) dose (2.4 mol. kg -1 L-[ring- 2 H 5 ]phenylalanine), after

3 doi: /jc jcem.endojournals.org 3 which a continuous L-[ring- 2 H 5 ]phenylalanine (0.06 mol. kg - 1.min -1 ) infusion was started. After the subjects rested in a supine position for 120 minutes, a second arterialized blood sample was drawn and a muscle biopsy was collected from the vastus lateralis muscle (t 0 minutes). Subjects then received a single bolus (250 ml) of test drink containing 20 g casein protein (NIZO, Ede, the Netherlands; see supplementary information). Thereafter, subjects rested in a semisupine position for another 240 minutes, during which arterialized blood samples were collected at regular intervals (t 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 minutes) after which a second muscle biopsy was collected (t 240 minutes), marking the end of the infusion period. Blood samples were collected into EDTA-containing tubes and centrifuged at 1000g for 10 minutes at 4 C. Aliquots of plasma were frozen in liquid nitrogen and stored at 80 C. The muscle biopsy samples were all taken from separate incisions from the leg identified as the leg to become immobilized (or the previously immobilized leg in the case of the second visit). Muscle biopsy samples were obtained from the middle region of the vastus lateralis, 15 cm above the patella and 3 cm below entry through the fascia, using the percutaneous needle biopsy technique (26). Muscle samples were dissected carefully and freed from any visible nonmuscle material and were immediately frozen in liquid nitrogen and stored at 80 C until further analysis. On the day of cast removal the experimental protocol was replicated exactly, so that postprandial muscle protein synthesis rates could be compared before and after immobilization. Limb immobilization Forty eight hours after the first stable isotope infusion test day, subjects attended the Casting Room at Maastricht University Medical Centre, at hours to have a full leg cast fitted to induce one-legged knee immobilization. The application of the cast signified the first day of the 14 day immobilization period. The circular leg cast extended from 10 cm above the ankle to approximately 25 cm above the patella. The knee was casted at a30 o angle of flexion to prevent subjects performing any weight bearing on the casted limb. Subjects were provided with crutches for proper ambulation (For complete details of casting procedure and materials please see supplementary information). All subjects were instructed to perform a series of simple ankle exercises (ie, plantar and dorsal flexion, and circular movements of the entire foot) to keep the calf muscle pump activated in the immobilized leg, thereby minimizing the risk of developing a deep vein thrombosis during the immobilization period. Subjects remained in the cast for 14 days, but were required to visit the Casting Room after 7 days to have the cast tightened. At the end of the 14 day immobilization period, subjects were collected from their home by car, and brought into the laboratory for the second stable isotope infusion experimental visit at hours. Prior to the start of the second test day, subjects visited the Casting Room to have the cast removed. Thereafter, subjects were taken by wheelchair to the laboratory to prevent any weight bearing, after which the stable isotope tracer experimental visit was repeated. Plasma analyses Plasma glucose and insulin concentrations were analyzed by Dr. Stein und Kollegen Laboratories (Mönchengladbach, Germany) using commercially available kits (GLUC3, Roche, Ref: , and Immunologic, Roche, Ref: , respectively). HbA1c content was determined in 3 ml venous blood samples by high-performance liquid chromatography (HPLC) (Bio-Rad Diamat, Munich, Germany). Plasma amino acid concentrations and enrichments were determined by GC-MS (Agilent 7890A GC/5975C; MSD, Little Falls, USA) as previously described (27) (see supplementary information). Muscle analyses L-[ring- 2 H 5 ]phenylalanine enrichment in mixed muscle protein and the muscle free pool were determined as previously described (27). Fractional rates of mixed muscle protein synthesis (FSR) were calculated by dividing the increment in enrichment in the product (ie, protein-bound L-[ring- 2 H 5 ]phenylalanine) by the enrichment of the precursor using the standard precursorproduct relationship as previously described (27, 28) (see supplementary information). Real time PCR and Western Blotting analyses were performed to determine mrna expression protein content, respectively, of key genes/signaling proteins in muscle samples. Phosphorylation status was expressed relative to the total amount of each protein (see supplementary information). Statistics All data are expressed as means SEM. A two-way ANOVA with time and visit (pre or post immobilization) as factors was used to compare differences in plasma glucose, insulin and amino acid concentrations, and plasma, muscle free and muscle protein bound L-[ring- 2 H 5 ]phenylalanine enrichments, and molecular signaling. When a significant interaction was detected, a Bonferonni post hoc test was applied to locate the individual differences. For all other variables, a paired t test was used to compare means before and after the immobilization period. Statistical significance was set at P.05. All calculations were performed by using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA). Results Habitual dietary intake Total daily energy intake averaged and MJ/d before and during the immobilization period (P.05 between conditions), with 45 3 and 46 2% of the energy provided by carbohydrate, 32 3 and 29 3% by fat, and 16 1 and 17 1% by protein, respectively. Total daily protein intake averaged and g/kg body mass/d before and during immobilization, respectively (P.05 between conditions). Muscle mass and strength Body mass, body mass index (BMI) and blood HbA 1c levels did not change over the experimental period (data not shown). Muscle quadriceps cross sectional area (CSA) determined by CT-scan of the midthigh is displayed in Figure 1. Quadriceps CSA did not differ between legs at baseline and remained unchanged throughout the experiment in the nonimmobilized leg. However, 2 weeks of leg immobilization resulted in an % decrease in

4 4 disuse and post-prandial muscle protein accretion J Clin Endocrinol Metab quadriceps muscle CSA (P.001). In agreement, leg lean mass assessed by DEXA was significantly reduced by % in the immobilized leg, representing a loss of g of muscle tissue (P.01), whereas in the control leg no changes were observed. Whole body lean and fat mass did not change throughout the experiment. The loss of muscle mass in the immobilized leg was accompanied by a 23 3% decline in leg muscle strength (from to kg; P.0001). No changes in muscle strength were observed in the nonimmobilized leg (from to kg; P.05). Plasma analyses Plasma glucose concentrations declined over time (P.001) during both experimental visits, but no differences were observed between visits (not shown). Plasma insulin concentrations (Figure 2A) showed a modest increase following protein ingestion up to 15 mu. L -1, after which concentrations returned to baseline levels (main effect of time; P.0001), although no differences were observed between visits. Both plasma leucine (Figure 2B) and phenylalanine (Figure 3A) concentrations increased (both P.0001 for time effect) following protein ingestion with no difference between visits. The time course of plasma L-[ring- 2 H 5 ]phenylalanine enrichments is illustrated in Figure 3B. From an enrichment of 8 mol per cent excess (MPE), the plasma tracer enrichment declined following protein ingestion (significant main effect of time; P.001), although no differences were observed between visits. Stable isotope analyses Mean muscle free and muscle protein-bound L-[ring- 2 H 5 ]phenylalanine enrichments and delta changes are presented in Table 1. Significant main effects for time (P.0001) and visit (P.05), and a significant time x visit interaction (P.05) were detected for mean muscle free L-[ring- 2 H 5 ]phenylalanine enrichments, with the fasted enrichment being lower (P.01) and a greater increase over time (P.05) both occurring post immobilization. Mean muscle protein bound L-[ring- 2 H 5 ]phenylalanine enrichments were higher in the fasted state post immobilization (P.001) and increased over time in both visits (P.0001) but to a greater extent in the pre immobilization visit (significant main effect for visit; P.0001, and interaction effect; P.05). These data translated into a mean change in muscle protein bound tracer enrichment over the 4 hours postprandial period of and in the pre and post immobilization visits, respectively (27 13% lower delta incorporation post Figure 1. Individual subject s quadriceps cross sectional area (CSA) before (PRE) and after (POST) 2 weeks of one-legged knee immobilization in the immobilized and nonimmobilized legs of healthy, young men (n 12). Numbers 1 12 denote individual subject s reference number. Data were analyzed with a two-way ANOVA (time x visit). In case of a significant interaction effect, Bonferroni post hoc tests were applied to locate the individual differences. Significant time (P.001) and interaction (P.001) effects. P.0001 comparing PRE and POST in the immobilized leg. Figure 2. Mean ( SEM) plasma insulin (A) and leucine (B) concentrations following ingestion of 20 g casein protein before (PRE) and after (POST) 2 weeks of one-legged knee immobilization in healthy, young men (n 12). Data were analyzed with a two-way ANOVA (time x visit). In case of a significant interaction effect, Bonferroni post hoc tests were applied to locate the individual differences. A: Significant time (P.0001) effect; interaction (P.361); treatment (P.835). B: significant time (P.0001) effect; interaction (P.686); treatment (P.564).

5 doi: /jc jcem.endojournals.org 5 Table 1. Tracer data Muscle free L-[ring- 2 H 5 ]phenylalanine enrichments Fasted PRE Fed *** change Fasted POST Fed ** *** change Muscle protein bound L-[ring- 2 H 5 ]phenylalanine enrichments Muscle free and protein bound L-[ring- 2 H 5 ]phenylalanine enrichments before (PRE) and after (POST) 2 weeks of leg immobilization and immediately before (Fasted) and 4 h after (Fed) the ingestion of 20 g dietary protein in healthy young men (n 12), and the change over the 4 h post-prandial period ( change). Enrichment expressed as mole per cent excess (MPE). For muscle free enrichments a significant main effect of time (P ), visit (P 0.05) and interaction (P 0.05). For muscle protein bound enrichments a significant main effect of time (P ), visit (P ) and interaction (P 0.05). Significant difference compared with corresponding PRE value: P 0.05, P Significant difference compared with corresponding Fasted value: ** P 0.01, *** P immobilization; P.05). Mixed muscle protein fractional synthetic rates (FSRs), with the weighted mean plasma (weighted to represent the average precursor labeling over time and to account for increased frequency of sampling over the first 60 minutes of the postprandial period; A) or mean muscle free (B) L-[ring- 2 H 5 ]phenylalanine enrichment as the precursor pool, are displayed in Figure 4. Mixed muscle FSR was 31 12% lower (P.05) in the post compared to pre immobilization visit ( and %. h -1, respectively), using the plasma precursor pool. Similar results were obtained when cal- Figure 3. Mean ( SEM) plasma phenylalanine concentrations (A) and L-[ring- 2 H 5 ]phenylalanine enrichments (B) following ingestion of 20 g casein protein before (PRE) and after (POST) 2 weeks of one-legged knee immobilization in healthy, young men (n 12). Data were analyzed with a two-way ANOVA (time x visit). In case of a significant interaction effect, Bonferroni post hoc tests were applied to locate the individual differences. A: significant time (P.0001) effect; interaction (P.745); treatment (P.828). B: significant time (P.0001) effect; interaction (P.659); treatment (P.385). Figure 4. Fractional muscle protein synthesis rates (FSR) using plasma (A) or muscle free (B) L-[ring- 2 H 5 ]phenylalanine enrichments as a precursor pool from 0 4 hours following the ingestion of 20 g casein protein before (PRE) and after (POST) 2 weeks of one-legged knee immobilization in healthy, young men (n 12). Data were analyzed with a paired t test. * significant difference between visits (P.05).

6 6 disuse and post-prandial muscle protein accretion J Clin Endocrinol Metab culating mixed muscle FSRs with the muscle free L-[ring- 2 H 5 ]phenylalanine enrichment as precursor, with a mean average of 50 22% lower FSRs in the post compared to pre immobilization visit ( and %. h -1, respectively; P.05). mrna expression and protein content Figure 5 depicts the muscle phosphorylation status of mammalian target of rapamycin (mtor; A) and p70 ribosomal protein S6 kinase (p70 S6K ; B) during both experimental visits. Data are expressed as the ratio of the phosphorylated protein to total protein content. There were no significant effects for either mtor or p70 S6K, of either protein ingestion (P.11 and P.67, respectively) or immobilization (P.34 and P.97, respectively). Figure 6 (supplementary information) presents the mrna expression of Muscle Atrophy F-Box/atrogin-1 (MAFbx; A), Muscle-Specific RING-finger protein 1 (MuRF1; B) and fork head box protein 01 (FOXO1; C)in baseline samples before and after the immobilization period. Muscle mrna expression of FOXO1 and MuRF1 Figure 5. Muscle phosphorylation status of mammalian target of rapamycin (mtor; A) and p70 ribosomal protein S6 kinase (p70 S6K ; B) before (PRE) and after (POST) 2 weeks of leg immobilization and immediately before (Fasted) and 4 hours after (Fed) the ingestion of 20 g casein protein in healthy young men (n 12) with representative blots for total and phosphorylated (p) forms of each protein (inset). Data are expressed as the ratio of the phosphorylated/total content of the selected proteins. Data were analyzed with a two-way ANOVA (time x visit). No significant main effects were detected. showed no changes with immobilization, while MAFbx expression had increased by 40%. Discussion The principal finding from the present study was that two weeks of one-legged knee immobilization impaired the muscle protein synthetic response to the ingestion of a 20 g bolus of dietary protein in healthy young men. As such, we provide data supportive of a key mechanistic role for anabolic resistance to meal ingestion in the development of muscle disuse atrophy. Muscle disuse leads to a rapid loss of muscle mass which brings with it an abundance of negative health consequences (16). As such, muscle disuse atrophy represents a significant health concern and forms an important area of clinical research. In the present study, two weeks of limb immobilization led to a 8.5% decrease (Figure 1) in cross sectional area of the quadriceps muscle. When quantified, this muscle atrophy equated to a loss of 350 g lean tissue from the immobilized leg. These data are in keeping with previous estimations that inactive muscle generally atrophies at a rate of around % per day during a short period of disuse (15, 16). We observed that this muscle loss was accompanied by a striking 25% loss of muscle strength, underlining the detrimental impact of muscle disuse on functional capacity. The physiological basis for disuse related muscle atrophy remains to be clearly elucidated. It has been well established that a decline in basal (fasting) muscle protein synthesis rates contributes to muscle loss observed during a period of disuse that exceeds 10 days (2, 5, 8 14), with little or no contribution of changes in muscle protein breakdown (2, 5, 11). Furthermore, recent work suggests that disuse reduces the sensitivity of skeletal muscle tissue to the anabolic properties of amino acids, which has been coined anabolic resistance (13, 21, 29, 30). The present study demonstrates that 14 days of immobilization induced a 30% reduction in the muscle protein synthetic response to the ingestion of 20 g of dietary casein protein (Figure 4). Thus, our data extend on previous findings, which used disuse models and amino acid administration (13, 21), by demonstrating that immobilization induces anabolic resistance under the physiological condition of ingesting a single meal-like bolus of protein in young adults. We carried out the present study under free living conditions, and monitored the habitual diet prior to and during the immobilization period. Daily energy intake and macronutrient composition of the diet did not differ between conditions, nor did the total daily protein intake (1.1 g/kg body mass/d before and during immobilization).

7 doi: /jc jcem.endojournals.org 7 Accordingly, we demonstrate that disuse leads to substantial muscle loss and declines in postprandial muscle protein synthesis rates under conditions where dietary protein consumption is maintained. Thus, compelling evidence now exists that, aside from changes in fasting muscle protein turnover, skeletal muscle protein synthesis becoming refractory to the normal stimulatory effects of protein ingestion contributes significantly to muscle disuse atrophy. Indeed, we know that the average baseline lean tissue mass in the immobilized leg of our volunteers was 12 kg. If we assume that habitual muscle protein synthesis rates are under stimulation of the last meal for 12 hours of each day (ie, 4 hours stimulation from each main meal) then it can be calculated that the present (disuse induced) decline in postprandial muscle protein synthesis would result in 11 g less muscle tissue synthesized per day. This equates to approximately 150 g less muscle protein synthesized over the 2 week immobilization period which accounts for over 40% of the observed muscle loss (ie, 347 g lean tissue). Anabolic resistance to dietary protein ingestion is now generally believed to play an important role in the development of age-related sarcopenia (31 34), as well as other situations associated with progressive muscle loss such as cancer cachexia (35), elective surgery (36, 37) and various critical illnesses (38, 39). Worthy of note, such conditions are all associated with decreased physical activity. In light of the present data, it is clear that disuse per se is capable of potently modulating the anabolic response to food intake. However, the underlying mechanism(s) responsible for anabolic resistance to protein intake remains to be established. Impairments may reside at the level of protein digestion (5, 27), amino acid absorption (5), postprandial hormonal response and subsequent microvascular perfusion (33, 40), muscle amino acid uptake (41) and/or intramuscular (IM) signaling (32, 42). In the present study, protein ingestion led to a rapid aminoacidemia, as evidenced by the significant increase in plasma leucine (Figure 2B) and phenylalanine (Figure 3A) concentrations. Furthermore, protein ingestion led to a dilution of the plasma L-[ring- 2 H 5 ]phenylalanine enrichment (Figure 3B). However, since none of the plasma amino acid or tracer responses differed between visits, it is unlikely that the digestion and absorption of the ingested protein was affected by the period of muscle disuse and thus did not contribute to the development of the observed anabolic resistance. Dietary protein ingestion also led to a similar plasma insulin response before and after immobilization (Figure 2A), suggesting that postprandial insulin mediated microvascular perfusion remained unaltered. However, it is also true that inactive muscle tissue quickly develops a reduced responsiveness to the physiological effects of insulin, at least regarding glucose uptake (43, 44). Moreover, it has been reported that anabolic resistance in older adults is associated with impaired insulin mediated vasodilation and thus amino acid supply (45). Whether disuse also blunts the postprandial response of the microvasculature to insulin remains to be seen, but may offer a potential mechanism whereby immobilization induces anabolic resistance. Recent data have suggested that the expression of key amino acid transporters in skeletal muscle may be a rate limiting step for postprandial amino acid uptake and subsequent muscle protein synthesis (41). It has been shown that 7 days of bed-rest reduces the muscle protein content of L-type amino acid transporter 1 (LAT1) and sodiumcoupled amino acid transporter 2 (SNAT2) (21). In the present study, following immobilization, a steady state in the muscle free tracer pool was not achieved prior to the first biopsy collection even with a primed, 2 hours tracer preinfusion period (Table 1). This would infer that basal, fasting amino acid transport into muscle tissue may have become impaired with disuse. However, of more relevance, the delta muscle free tracer enrichment over the 4 hours postprandial period was 3 fold higher following immobilization (Table 1) which is not consistent with a reduced amino acid uptake in response to protein ingestion and hyperinsulinemia. It could be speculated that muscle protein breakdown was reduced following immobilization, meaning that less unlabeled phenylalanine efflux was diluting the muscle free L-[ring- 2 H 5 ]phenylalanine pool. We assessed the gene expression of two primary ubiquitin ligases, Muscle Atrophy F-Box/ atrogin-1 (MAFbx) and Muscle-Specific RING-finger protein 1 (MuRF1), and their key transcription factor, fork head box protein 01 (FOXO1) (46) to obtain some insight in the possibility of changes in muscle protein breakdown (Figure 6, supplementary information). However, in line with previous work (5, 11) we were unable to find any evidence suggesting a decline in muscle protein breakdown following immobilization. Rather than changes in amino acid uptake or rates of muscle protein breakdown, a more likely explanation for the accumulation of muscle free tracer post immobilization may simply be a reduced ability of the muscle cell to incorporate the available amino acids into new muscle proteins (Table 1). If so, this would suggest that the mechanism(s) underlying the observed anabolic resistance is intracellular. Acute changes in muscle protein synthesis rates are regulated at the post-translational level by changes in the phosphorylation status of key IM signaling proteins; notably the mammalian target of rapamycin (mtor) and its subsequent downstream activation of P70 S6 protein kinase (p70 S6K ) (47, 48). In support of previous studies (13,

8 8 disuse and post-prandial muscle protein accretion J Clin Endocrinol Metab 14), we provide evidence that 2 weeks of limb immobilization does not alter the phosphorylation status of either mtor or p70 S6K in the basal state (Figure 5). Furthermore, we observed no significant increase in either mtor or p70 S6K phosphorylation following dietary protein ingestion either before or after immobilization and, thus, did not reveal any evidence for altered anabolic signaling with disuse. Previous studies have reported an increase in phosphorylation status of mtor and/or p70 S6K following protein/amino acid ingestion (eg, 21, 49). Peak stimulation of these signaling proteins generally occurs 1 2 hours following protein ingestion (21, 49 51), with such effects being greatly attenuated or no longer present after 3 6 hours (21, 49 51). The presented data provide a single snapshot taken 4 hours after protein ingestion, where rapid, transient changes may no longer persist. Future research studies aiming to elucidate the molecular mechanisms underpinning this anabolic resistance will require multiple biopsy sampling points across several hours. In the present study, we applied limb immobilization as an experimental model to examine the impact of disuse on muscle tissue in the absence of any major systemic adaptations as seen following prolonged bed-rest. However, it should be noted that the term disuse describes a spectrum of behavior, from reduced habitual physical activity (ie, sedentary living) through to whole body disuse. Interestingly, data are now accumulating to show that the loss of muscle mass and strength (52, 53), and an associated reduction in anabolic sensitivity to protein (53), also occurs following merely two weeks of reduced physical activity (decreased step count). Thus, regardless of the severity of experimental disuse applied, anabolic resistance to dietary protein represents an important mechanism underlying muscle disuse atrophy. As such, from a research and/or clinical perspective, differing forms of disuse should be considered together as part of a continuum. From a clinical nutrition perspective, it has been proposed that increasing the amount of dietary protein/essential amino acids in the diet represents an effective means to attenuate muscle disuse atrophy (10, 16, 54, 55). Whereas some studies report an attenuation of muscle disuse atrophy with increased protein/amino acid intake (10, 16, 54, 55), others have failed to confirm these findings (56 58). Interestingly, the development of anabolic resistance to a normal protein bolus may partly explain the lack of consistency between these studies. Specifically, dietary interventions that can compensate for disuse induced anabolic resistance need to maximize the muscle protein synthetic response to food intake. Such strategies may include increasing the amount of dietary protein consumed (59, 60), supplementing essential amino acids (32, 54), using more rapidly digestible protein sources (27, 50, 61, 62), fortifying dietary protein with free leucine (63 65) and/or altering the timing of protein ingestion (66). Such nutritional strategies should be evaluated for their clinical efficacy to attenuate the loss of muscle mass and function during short as well as longer periods of disuse. In conclusion, two weeks of muscle disuse leads to a reduced muscle protein synthetic response to the ingestion of a meal-like quantity of dietary protein. Accordingly, anabolic resistance to food intake plays an important mechanistic role in muscle disuse atrophy. Acknowledgments We gratefully acknowledge the enthusiastic support of the entire team from the Maastricht University Hospital Casting Room, Dr Stein Laboratories for analyzing the plasma insulin and glucose concentrations, Rinske Franssen for dietary analyses, and the volunteers for their participation in the study. BTW and LJCvL designed the study. BTW and TS organized and carried out the clinical experiments. APG performed the mass spectrometry and amino acid analyses. JMGS performed the western blot analyses. BTW performed the real time PCR analyses. BTW performed the statistical analysis of the data and wrote the manuscript together with LJCvL. All the authors contributed to the interpretation of the data and approved the final version of the manuscript. None of the authors had a personal or financial conflict of interest. Address all correspondence and requests for reprints to: Corresponding author: Luc JC van Loon, PhD, Department of Human Movement Sciences, Maastricht University Medical Centre, Maastricht, 6200 MD, the Netherlands, Tel: 31 (0) , l.vanloon@maastrichtuniversity.nl. No funding sources to declare. This work was supported by. References 1. Deitrick J. The effect of immobilization on metabolic and physiological functions of normal men. Bull N Y Acad Med. 1948;24: Gibson J, Halliday D, Morrison W, Stoward P, Hornsby G, Watt P, Murdoch G, Rennie M. Decrease in human quadriceps muscle protein turnover consequent upon leg immobilization. Clin Sci (Lond). 1987;72: Stuart C, Shangraw R, Prince M, Peters E, Wolfe R. Bed-rest-induced insulin resistance occurs primarily in muscle. Metabolism. 1988;37: Haruna Y, Suzuki Y, Kawakubo K, Yanagibori R, Gunji A. Decremental reset in basal metabolism during 20-days bed rest. 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