Loss of Muscle Mass and Strength After Hip Fracture: an Intervention Target for Nutrition Supplementation.

PURPOSEOF REVIEW: To summarize what is known about the deleterious effect of hip fracture on muscle mass and strength as well as the scientific evidence for post-surgical nutrition supplementation to maintain muscle and improve function. RECENT FINDINGS: This review provides a discussion of the relationship between muscle mass, strength, and physical function following hip fracture, briefly describes the approaches to measuring lean mass, discusses prevalence of sarcopenia and malnutrition among older men and women with hip fracture, and reviews the effects of essential amino acids on muscle. Loss of muscle mass and strength following hip fracture is substantial with consequences for recovery of functional independence. EAA-based nutrition supplementation, which directly effects muscle, has potential to improve outcomes.

Essential amino acid supplementation alters the p53 transcriptional response and cytokine gene expression following total knee arthroplasty.

Reducing muscle atrophy following orthopedic surgery is critical during the postoperative period. Our previous work in patients who underwent total knee arthroplasty (TKA) showed that the vast majority of atrophy occurs within 2 wk following surgery and that essential amino acid (EAA) supplementation attenuates this atrophy. We used RNA-sequencing (RNA-seq) to identify genes associated with atrophy after TKA with and without EAAs. Analysis of overrepresented gene-ontology terms revealed that p53 signaling and the cytokine-cytokine receptor pathways were highly upregulated after TKA. Relative to the placebo group, the EAA group had altered expression of p53 regulators such as MDM2. This altered expression may account for differences between groups in timing of upregulation of some p53 targets such as apoptosis genes, and may account for the reduction in muscle loss in the subjects receiving EAAs. Furthermore, we observed altered expression of a large number of cytokine-signaling genes including TNFRSF12A, which plays a critical role in muscle atrophy, myogenesis, fibrosis, and the noncanonical NF-κB pathway.NEW & NOTEWORTHY Total knee arthroplasty is the most frequently performed inpatient surgical procedure for those over 45 yr in the United States. Following surgery, patients lose a large amount of muscle, which impacts functional mobility. Previously, our laboratory found that supplementing patients' diets with essential amino acids (EAAs) reduces postsurgical muscle loss. Here, our goal was to characterize the transcriptional changes associated with surgery with and without EAA supplementation to uncover the underlying mechanisms by which EAAs attenuate this muscle loss.

Cellular and morphological changes with EAA supplementation before and after total knee arthroplasty.

Investigate the underlying cellular basis of muscle atrophy (Placebo) and atrophy reduction (essential amino acid supplementation, EAAs) in total knee arthroplasty (TKA) patients by examining satellite cells and other key histological markers of inflammation, recovery, and fibrosis. Forty-one subjects (53-76 yr) scheduled for TKA were randomized into two groups, ingesting 20 g of EAAs or placebo, twice-daily, for 7 days before TKA and for 6 wk after surgery. A first set of muscle biopsies was obtained from both legs before surgery in the operating room, and patients were randomly assigned and equally allocated to have two additional biopsies at either 1 or 2 wk after surgery. Biopsies were processed for gene expression and immunohistochemistry. Satellite cells were significantly higher in patients ingesting 20 g of essential amino acids twice daily for the 7 days leading up to surgery compared with Placebo (operative leg P = 0.03 for satellite cells/fiber and P = 0.05 for satellite cell proportions for Type I-associated cells and P = 0.05 for satellite cells/fiber for Type II-associated cells.) Myogenic regulatory factor gene expression was different between groups, with the Placebo Group having elevated MyoD expression at 1 wk and EAAs having elevated myogenin expression at 1 wk. M1 macrophages were more prevalent in Placebo than the EAAs Group. IL-6 and TNF-α transcripts were elevated postsurgery in both groups; however, TNF-α declined by 2 wk in the EAAs Group. EAAs starting 7 days before surgery increased satellite cells on the day of surgery and promoted a more favorable inflammatory environment postsurgery.NEW & NOTEWORTHY Clinical studies by our group indicate that the majority of muscle atrophy after total knee arthroplasty (TKA) in older adults occurs rapidly, within the first 2 wks. We have also shown that essential amino acid supplementation (EAAs) before and after TKA mitigates muscle atrophy; however, the mechanisms are unknown. These results suggest that satellite cell numbers are elevated with EAA ingestion before surgery, and after surgery, EAA ingestion positively influences markers of inflammation. Combined, these data may help inform further studies designed to address the accelerated sarcopenia that occurs in older adults after major surgery.

Essential Amino Acid Supplementation Mitigates Muscle Atrophy After Total Knee Arthroplasty: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Substantial muscle atrophy occurs after total knee arthroplasty (TKA), resulting in decreased strength and impaired mobility. We sought to determine whether perioperative supplementation with essential amino acids (EAA) would attenuate muscle atrophy following TKA and whether the supplements were safe for ingestion in an older surgical population. METHODS: We performed a double-blind, placebo-controlled, randomized trial of 39 adults (age range, 53 to 76 years) undergoing primary unilateral TKA who ingested 20 g of EAA (n = 19) or placebo (n = 20) twice daily for 7 days preoperatively and for 6 weeks postoperatively. At baseline and 6 weeks postoperatively, magnetic resonance imaging (MRI) scans were obtained to measure quadriceps and hamstrings muscle volume. Secondary outcomes included functional mobility and strength. Data on physical activity, diet, and patient-reported outcomes (Veterans RAND 12-Item Health Survey and Knee injury and Osteoarthritis Outcome Score) were collected. Safety was determined through blood tests evaluating blood urea nitrogen, creatinine, creatinine clearance, homocysteine, and renal and liver function. Laboratory values at baseline, on the day of surgery, and at 2 days, 2 weeks, and 6 weeks postoperatively were compared between treatment groups. Analysis of covariance models, with baseline values as covariates, were used to evaluate outcomes between treatment groups. P values were adjusted for multiple tests. RESULTS: Compared with baseline, the EAA group had significantly less decrease in mean quadriceps muscle volume compared with the placebo group in the involved leg (-8.5% ± 2.5% compared with -13.4% ± 1.9%; p = 0.033) and the contralateral leg (-1.5% ± 1.6% compared with -7.2% ± 1.4%; p = 0.014). The hamstrings also demonstrated a greater muscle-volume-sparing effect for the EAA group than for the placebo group in the involved leg (-7.4% ± 2.0% compared with -12.2% ± 1.4%; p = 0.036) and contralateral leg (-2.1% ± 1.3% compared with -7.5% ± 1.5%; p = 0.005). There were no differences between the groups in terms of functional measures or strength. Blood chemistry values varied significantly between assessments periods but did not statistically differ between groups. CONCLUSIONS: The results of the present study suggest that EAA supplementation is safe and reduces the loss of muscle volume in older adults recovering from TKA. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Adaptive remodeling of skeletal muscle energy metabolism in high-altitude hypoxia: Lessons from AltitudeOmics.

Metabolic responses to hypoxia play important roles in cell survival strategies and disease pathogenesis in humans. However, the homeostatic adjustments that balance changes in energy supply and demand to maintain organismal function under chronic low oxygen conditions remain incompletely understood, making it difficult to distinguish adaptive from maladaptive responses in hypoxia-related pathologies. We integrated metabolomic and proteomic profiling with mitochondrial respirometry and blood gas analyses to comprehensively define the physiological responses of skeletal muscle energy metabolism to 16 days of high-altitude hypoxia (5260 m) in healthy volunteers from the AltitudeOmics project. In contrast to the view that hypoxia down-regulates aerobic metabolism, results show that mitochondria play a central role in muscle hypoxia adaptation by supporting higher resting phosphorylation potential and enhancing the efficiency of long-chain acylcarnitine oxidation. This directs increases in muscle glucose toward pentose phosphate and one-carbon metabolism pathways that support cytosolic redox balance and help mitigate the effects of increased protein and purine nucleotide catabolism in hypoxia. Muscle accumulation of free amino acids favor these adjustments by coordinating cytosolic and mitochondrial pathways to rid the cell of excess nitrogen, but might ultimately limit muscle oxidative capacity in vivo Collectively, these studies illustrate how an integration of aerobic and anaerobic metabolism is required for physiological hypoxia adaptation in skeletal muscle, and highlight protein catabolism and allosteric regulation as unexpected orchestrators of metabolic remodeling in this context. These findings have important implications for the management of hypoxia-related diseases and other conditions associated with chronic catabolic stress.

Evidence of a broad histamine footprint on the human exercise transcriptome.

KEY POINTS: Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors, in a variety of pathways that probably predate its more recent role in innate and adaptive immunity. Although histamine is normally associated with pathological conditions or allergic and anaphylactic reactions, it may contribute beneficially to the normal changes that occur within skeletal muscle during the recovery from exercise. We show that the human response to exercise includes an altered expression of thousands of protein-coding genes, and much of this response appears to be driven by histamine. Histamine may be an important molecular transducer contributing to many of the adaptations that accompany chronic exercise training. ABSTRACT: Histamine is a primordial signalling molecule, capable of activating cells in an autocrine or paracrine fashion via specific cell surface receptors. In humans, aerobic exercise is followed by a post-exercise activation of histamine H1 and H2 receptors localized to the previously exercised muscle. This could trigger a broad range of cellular adaptations in response to exercise. Thus, we exploited RNA sequencing to explore the effects of H1 and H2 receptor blockade on the exercise transcriptome in human skeletal muscle tissue harvested from the vastus lateralis. We found that exercise exerts a profound influence on the human transcriptome, causing the differential expression of more than 3000 protein-coding genes. The influence of histamine blockade post-exercise was notable for 795 genes that were differentially expressed between the control and blockade condition, which represents >25% of the number responding to exercise. The broad histamine footprint on the human exercise transcriptome crosses many cellular functions, including inflammation, vascular function, metabolism, and cellular maintenance.

Tourniquet Use During Knee Replacement Surgery May Contribute to Muscle Atrophy in Older Adults.

Muscle atrophy after total knee arthroplasty (TKA) occurs at a rate of 1% per day for the first 2 wk. Our hypothesis is that tourniquet-induced ischemia-reperfusion injury occurring during TKA influences metabolism and may contribute to atrophy. Identifying pathways that are upregulated during this critical "14-d window" after surgery may help us delineate therapeutic approaches to avoid muscle loss.

Transcriptional profiling and muscle cross-section analysis reveal signs of ischemia reperfusion injury following total knee arthroplasty with tourniquet.

Total knee arthroplasty (TKA) is the most common and cost-effective treatment for older adults with long-standing osteoarthritis. Projections indicate that nearly 3.5 million older adults will undergo this procedure annually by the year 2030. Thus, understanding the factors that lead to optimal outcomes is of great clinical interest. In the majority of cases, tourniquet is applied during surgery to maintain a clear surgical field, however, there is debate as to whether this intervention is completely benign. In particular, muscle atrophy is a significant factor in preventing full functional recovery following surgery, and some evidence suggests that tourniquet application and the associated ischemia-reperfusion injury that results contributes to muscle atrophy. For this reason, we examined tissue level changes in muscle in TKA patients following surgery and found that there was a significant increase in cross-sectional area of muscle fibers of all types. Furthermore, to detect changes not evident at the tissue level, we performed NextSeq analysis to assess the transcriptional landscape of quadriceps muscle cells following TKA with tourniquet and found 72 genes that were significantly upregulated. A large proportion of those genes regulate cell stress pathways, suggesting that muscle cells in our cohort of older adults were capable of mounting a significant response to cell stress. Furthermore, factors related to complement were upregulated, suggesting tourniquet may play a role in priming cells to ischemia reperfusion injury. Therefore, our analysis reveals potential harms of tourniquet during TKA, thus suggesting that surgeons should consider limiting its use.

Essential amino acid supplementation in patients following total knee arthroplasty.

BACKGROUND: By the year 2030, 3.48 million older U.S. adults are projected to undergo total knee arthroplasty (TKA). Following this surgery, considerable muscle atrophy occurs, resulting in decreased strength and impaired functional mobility. Essential amino acids (EAAs) have been shown to attenuate muscle loss during periods of reduced activity and may be beneficial for TKA patients. METHODS: We used a double-blind, placebo-controlled, randomized clinical trial with 28 older adults undergoing TKA. Patients were randomized to ingest either 20 g of EAAs (n = 16) or placebo (n = 12) twice daily between meals for 1 week before and 2 weeks after TKA. At baseline, 2 weeks, and 6 weeks after TKA, an MRI was performed to determine mid-thigh muscle and adipose tissue volume. Muscle strength and functional mobility were also measured at these times. RESULTS: TKA patients receiving placebo exhibited greater quadriceps muscle atrophy, with a -14.3 ± 3.6% change from baseline to 2 weeks after surgery compared with -3.4 ± 3.1% for the EAA group (F = 5.16, P = 0.036) and a -18.4 ± 2.3% change from baseline to 6 weeks after surgery for placebo versus -6.2 ± 2.2% for the EAA group (F = 14.14, P = 0.001). EAAs also attenuated atrophy in the nonoperated quadriceps and in the hamstring and adductor muscles of both extremities. The EAA group performed better at 2 and 6 weeks after surgery on functional mobility tests (all P < 0.05). Change in quadriceps muscle atrophy was significantly associated with change in functional mobility (F = 5.78, P = 0.021). CONCLUSION: EAA treatment attenuated muscle atrophy and accelerated the return of functional mobility in older adults following TKA. TRIAL REGISTRATION: Clinicaltrials.gov NCT00760383.

Endoplasmic Reticulum Stress Activation during Total Knee Arthroplasty.

Total knee arthroplasty (TKA) is the most common remediation for knee pain from osteoarthritis (OA) and is performed 650,000 annually in the U.S. A tourniquet is commonly used during TKA which causes ischemia and reperfusion (I/R) to the lower limb but the effects of I/R on muscle are not fully understood. Previous reports suggest upregulation of cell-stress and catabolism and downregulation of markers of cap-dependent translation during and after TKA. I/R has also been shown to cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). We hypothesized that the UPR would be activated in response to ER stress during TKA. We obtained muscle biopsies from the vastus lateralis at baseline, before TKA; at maximal ischemia, prior to tourniquet deflation; and during reperfusion in the operating room. Phosphorylation of 4E-BP1 and AKT decreased during ischemia (-28%, p < .05; -20%, p < .05 respectively) along with an increase in eIF2α phosphorylation (64%, p < .05) suggesting decreased translation initiation. Cleaved ATF6 protein increased in ischemia (39%, p = .056) but returned to baseline during reperfusion. CASP3 activation increased during reperfusion compared to baseline (23%, p < .05). XBP1 splicing assays revealed an increase in spliced transcript during ischemia (31%, p < .05) which diminished during reperfusion. These results suggest that in response to I/R during TKA all three branches of the ER stress response are activated.

AICAR administration ameliorates hypertension and angiogenic imbalance in a model of preeclampsia in the rat.

Previous studies suggest restoration of angiogenic balance can lower blood pressure and improve vascular endothelium function in models of preeclampsia. Our laboratory has recently reported exercise training mitigates hypertension in an animal model of preeclampsia, but the mechanisms are unknown. AMP-activated protein kinase (AMPK) is stimulated during exercise and has been shown to increase expression of VEGF. Therefore, the purpose of this study was to determine whether AICAR (5-aminoimidazole-4-carboxamide-3-ribonucleoside), a potent AMPK stimulator, would increase circulating VEGF, improve angiogenic potential, decrease oxidative stress, and abrogate placental ischemia-induced hypertension. In rats, reduced uteroplacental perfusion pressure (RUPP) was induced on day 14 of gestation by introducing silver clips on the inferior abdominal aorta and ovarian arteries. AICAR was administered intraperitoneally (50 mg/kg b.i.d.) days 14-18, and blood pressure and tissues were collected on day 19. RUPP-induced hypertension was ameliorated (P < 0.05) with AICAR versus RUPP. AICAR increased (P < 0.05) plasma VEGF and decreased (P < 0.05) plasma soluble VEGF receptor-1 in the RUPP + AICAR versus RUPP. Antioxidant capacity was restored (P < 0.05) by AICAR in RUPP placenta. Renal and placental catalase activity was decreased (P < 0.05) in RUPP + AICAR versus RUPP. Angiogenic potential was increased (P < 0.05) in RUPP + AICAR versus RUPP. Fetal and placental weights were unaffected by AICAR. Placental AMPK phosphorylation was increased (P < 0.05) in RUPP + AICAR versus normal pregnant and RUPP. These findings suggest AICAR may be useful to mitigate angiogenic imbalance, renal, and placental oxidative stress and increase in blood pressure associated with RUPP hypertension. Furthermore, placental AMPK phosphorylation was observed only in the setting of ischemia.

Placental and vascular adaptations to exercise training before and during pregnancy in the rat.

Although exercise during pregnancy is generally recommended and thought to be beneficial to mother and fetus, the nature of the adaptations to exercise during pregnancy and how they may be beneficial remain poorly understood. Recent studies suggest that exercise may stimulate expression of several cytoprotective and pro-angiogenic molecules such as heat shock proteins (HSP) and vascular endothelial growth factors (VEGF). We hypothesized that exercise training during pregnancy improves angiogenic balance, increases HSP expression, and improves endothelial function. Female rats were given access to an exercise wheel for 6 wk before and during pregnancy. On day 19 of pregnancy tissues were collected and snap frozen for later analysis. Western blots were performed in skeletal muscle and placenta. HSP 27 (3.7 ± 0.36 vs. 2.2 ± 0.38; P < 0.05), HSP 60 (2.2 ± 0.73 vs. 0.49 ± 0.08; P < 0.05), and HSP 90 (0.33 ± 0.09 vs. 0.11 ± 0.02; P < 0.05) were increased in the placentas of exercise-trained rats compared with sedentary controls. In addition, exercise training increased (P < 0.05) plasma free VEGF and augmented (P < 0.05) endothelium-dependent vascular relaxation compared with nonexercise control rats. The present data indicates chronic exercise training stimulates HSP expression in the placenta and that regular exercise training increases circulating VEGF in pregnant but not in nonpregnant rats. Although the present findings suggest that exercise before and during pregnancy may promote the expression of molecules that could attenuate placental and vascular dysfunction in complicated pregnancies, further studies are needed to determine the safety and effectiveness of exercise training as a therapeutic modality in pregnancy.

MAFbx, MuRF1, and the stress-activated protein kinases are upregulated in muscle cells during total knee arthroplasty.

Total knee arthroplasty (TKA) is the most common and a cost-effective surgical remediation for older adults with long-standing osteoarthritis. In parallel with the expanding population of older adults, the number of TKAs performed annually is projected to be 3.48 million by 2030. During this surgery, a tourniquet is used to stop blood flow to the operative leg. However, the molecular pathways that are affected by tourniquet use during TKA continue to be elucidated. We hypothesized that components of the catabolic FoxO3a (i.e., MuRF1, MAFbx, and Bnip3) pathway, as well as the cellular stress pathways [i.e., stress-activated protein kinase (SAPK)/JNK and MAPKs], are upregulated during TKA. The purpose of this study was to measure changes in transcripts and proteins involved in muscle cell catabolic and stress-activated pathways. We obtained muscle biopsies from subjects, 70 ± 1.3 yr, during TKA, from the vastus lateralis at baseline (before tourniquet inflation), during maximal ischemia (just before tourniquet release), and during reperfusion. Total tourniquet time was 43 ± 2 min and reperfusion time was 16 ± 1. Significant increases in FoxO3a downstream targets, MAFbx and MuRF1, were present for mRNA levels during ischemia (MAFbx, P = 0.04; MuRF1, P = 0.04), and protein expression during ischemia (MAFbx, P = 0.002; MuRF1, P = 0.001) and reperfusion (MuRF1, P = 0.002). Additionally, stress-activated JNK gene expression (P = 0.01) and protein were elevated during ischemia (P = 0.001). The results of this study support our hypothesis that protein degradation pathways are stimulated during TKA. Muscle protein catabolism is likely to play a role in the rapid loss of muscle volume measured within 2 wk of this surgery.

Proteins regulating cap-dependent translation are downregulated during total knee arthroplasty.

Total knee arthroplasty (TKA) utilizes a tourniquet to reduce blood loss, maintain a clear surgical "bloodless" field, and to ensure proper bone-implant cementing. In 2007, over 600,000 TKAs were performed in the United States, and this number is projected to increase to 3.48 million procedures performed annually by 2030. The acute effects of tourniquet-induced ischemia-reperfusion (I/R) on human skeletal muscle cells are poorly understood and require critical investigation, as muscle atrophy following this surgery is rapid and represents the most significant clinical barrier to long-term normalization of physical function. To determine the acute effects of I/R on skeletal muscle cells, biopsies were obtained at baseline, maximal ischemia (prior to tourniquet release), and reperfusion (following tourniquet release). Quadriceps volume was determined before and 2 wk post-TKA by MRI. We measured a 36% decrease in phosphorylation of Akt Ser(473) during ischemia and 37% during reperfusion (P < 0.05). 4E-BP1 Thr(37/46) phosphorylation decreased 29% during ischemia and 22% during reperfusion (P < 0.05). eEF2 Thr(56) phosphorylation increased 25% during ischemia and 43% during reperfusion (P < 0.05). Quadriceps volume decreased 12% in the TKA leg (P < 0.05) and tended to decrease (6%) in the contralateral leg (P = 0.1). These data suggest cap-dependent translation initiation, and elongation may be inhibited during and after TKA surgery. We propose that cap-dependent translational events occurring during surgery may precipitate postoperative changes in muscle cells that contribute to the etiology of muscle atrophy following TKA.

Pharmacological vasodilation improves insulin-stimulated muscle protein anabolism but not glucose utilization in older adults.

OBJECTIVE: Skeletal muscle protein metabolism is resistant to the anabolic action of insulin in healthy, nondiabetic older adults. This defect is associated with impaired insulin-induced vasodilation and mTORC1 signaling. We hypothesized that, in older subjects, pharmacological restoration of insulin-induced capillary recruitment would improve the response of muscle protein synthesis and anabolism to insulin. RESEARCH DESIGN AND METHODS: Twelve healthy, nondiabetic older subjects (71 ± 2 years) were randomized to two groups. Subjects were studied at baseline and during local infusion in one leg of insulin alone (Control) or insulin plus sodium nitroprusside (SNP) at variable rate to double leg blood flow. We measured leg blood flow by dye dilution; muscle microvascular perfusion with contrast enhanced ultrasound; Akt/mTORC1 signaling by Western blotting; and muscle protein synthesis, amino acid, and glucose kinetics using stable isotope methodologies. RESULTS: There were no baseline differences between groups. Blood flow, muscle perfusion, phenylalanine delivery to the leg, and intracellular availability of phenylalanine increased significantly (P < 0.05) in SNP only. Akt phosphorylation increased in both groups but increased more in SNP (P < 0.05). Muscle protein synthesis and net balance (nmol · min(-1) · 100 ml · leg(-1)) increased significantly (P < 0.05) in SNP (synthesis, 43 ± 6 to 129 ± 25; net balance, -16 ± 3 to 26 ± 12) but not in Control (synthesis, 41 ± 10 to 53 ± 8; net balance, -17 ± 3 to -2 ± 3). CONCLUSIONS: Pharmacological enhancement of muscle perfusion and amino acid availability during hyperinsulinemia improves the muscle protein anabolic effect of insulin in older adults.

Muscle protein breakdown has a minor role in the protein anabolic response to essential amino acid and carbohydrate intake following resistance exercise.

Muscle protein breakdown (MPB) is increased following resistance exercise, but ingestion of carbohydrate during postexercise recovery can decrease MPB with no effect on muscle protein synthesis (MPS). We sought to determine whether a combination of essential amino acids (EAA) with low carbohydrate or high carbohydrate could effectively reduce MPB following resistance exercise and improve muscle protein net balance (NB). We hypothesized that higher levels of carbohydrate and resulting increases in circulating insulin would inhibit MPB and associated signaling, resulting in augmented NB. Thirteen male subjects were assigned to one of two groups receiving equivalent amounts of EAA (approximately 20 g) but differing carbohydrate levels (low = 30, high = 90 g). Groups ingested nutrients 1 h after an acute bout of leg resistance exercise. Leg phenylalanine kinetics (e.g., MPB, MPS, NB), signaling proteins, and mRNA expression were assessed on successive muscle biopsies using stable isotopic techniques, immunoblotting, and real-time quantitative PCR, respectively. MPB tended to decrease (P < 0.1) and MPS increased (P < 0.05) similarly in both groups following nutrient ingestion. No group differences were observed, but muscle ring finger 1 (MuRF1) protein content and MuRF1 mRNA expression increased following resistance exercise and remained elevated following nutrient ingestion, while autophagy marker (light-chain 3B-II) decreased after nutrient ingestion (P < 0.05). Forkhead box-O3a phosphorylation, total muscle atrophy F-box (MAFbx) protein, and MAFbx and caspase-3 mRNA expression were unchanged. We conclude that the enhanced muscle protein anabolic response detected when EAA+carbohydrate are ingested postresistance exercise is primarily due to an increase in MPS with minor changes in MPB, regardless of carbohydrate dose or circulating insulin level.

Insulin stimulates human skeletal muscle protein synthesis via an indirect mechanism involving endothelial-dependent vasodilation and mammalian target of rapamycin complex 1 signaling.

OBJECTIVE: Our objective was to determine whether endothelial-dependent vasodilation is an essential mechanism by which insulin stimulates human skeletal muscle protein synthesis and anabolism. SUBJECTS: Subjects were healthy young adults (n=14) aged 31+/-2 yr. DESIGN: Subjects were studied at baseline and during local leg infusion of insulin alone (control, n=7) or insulin plus the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, n=7) to prevent insulin-induced vasodilation. METHODS: We measured skeletal muscle protein metabolism with stable isotope tracers, blood flow with indocyanine green, capillary recruitment with contrast enhanced ultrasound, glucose metabolism with stable isotope tracers, and phosphorylation of proteins associated with insulin (Akt) and amino acid-induced mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling (mTOR, S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1) with Western blot analysis. RESULTS: No basal differences between groups were detected. During insulin infusion, blood flow and capillary recruitment increased in the control (P<0.05) group only; Akt phosphorylation and glucose uptake increased in both groups (P<0.05), with no group differences; and mTORC1 signaling increased more in control (P<0.05) than in L-NMMA. Phenylalanine net balance increased (P<0.05) in both groups, but with opposite mechanisms: increased protein synthesis (basal, 0.051+/-0.006 %/h; insulin, 0.077+/-0.008 %/h; P<0.05) with no change in proteolysis in control and decreased proteolysis (P<0.05) with no change in synthesis (basal, 0.061+/-0.004 %/h; insulin, 0.050+/-0.006 %/h; P value not significant) in L-NMMA. CONCLUSIONS: Endothelial-dependent vasodilation and the consequent increase in nutritive flow and mTORC1 signaling, rather than Akt signaling, are fundamental mechanisms by which insulin stimulates muscle protein synthesis in humans. Additionally, these data underscore that insulin modulates skeletal muscle proteolysis according to its effects on nutritive flow.

Rapamycin administration in humans blocks the contraction-induced increase in skeletal muscle protein synthesis.

Muscle protein synthesis and mTORC1 signalling are concurrently stimulated following muscle contraction in humans. In an effort to determine whether mTORC1 signalling is essential for regulating muscle protein synthesis in humans, we treated subjects with a potent mTORC1 inhibitor (rapamycin) prior to performing a series of high-intensity muscle contractions. Here we show that rapamycin treatment blocks the early (1-2 h) acute contraction-induced increase ( approximately 40%) in human muscle protein synthesis. In addition, several downstream components of the mTORC1 signalling pathway were also blunted or blocked by rapamycin. For instance, S6K1 phosphorylation (Thr421/Ser424) was increased post-exercise 6-fold in the control group while being unchanged with rapamycin treatment. Furthermore, eEF2 phosphorylation (Thr56) was reduced by approximately 25% post-exercise in the control group but phosphorylation following rapamycin treatment was unaltered, indicating that translation elongation was inhibited. Rapamycin administration prior to exercise also reduced the ability of raptor to associate with mTORC1 during post-exercise recovery. Surprisingly, rapamycin treatment prior to resistance exercise completely blocked the contraction-induced increase in the phosphorylation of ERK1/2 (Thr202/Tyr204) and blunted the increase in MNK1 (Thr197/202) phosphorylation. However, the phosphorylation of a known target of MNK1, eIF4E (Ser208), was similar in both groups (P > 0.05) which is consistent with the notion that rapamycin does not directly inhibit MAPK signalling. We conclude that mTORC1 signalling is, in part, playing a key role in regulating the contraction-induced stimulation of muscle protein synthesis in humans, while dual activation of mTORC1 and ERK1/2 stimulation may be required for full stimulation of human skeletal muscle protein synthesis.