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Ovarian cancer (OVC) is one of the most aggressive gynecological malignancies worldwide. Although olaparib treatment has shown favorable outcomes against the treatment of OVC, its effectiveness remains limited in some OVC patients. Investigating new strategies to improve the therapeutic efficacy of olaparib against OVC is imperative. Our study identified tabersonine, a natural indole alkaloid, for its potential to increase the chemosensitivity of olaparib in OVC. The combined treatment of olaparib and tabersonine synergistically inhibited cell proliferation in OVC cells and suppressed tumor growth in A2780 xenografts. The combined treatment effectively suppressed epithelial-mesenchymal transition (EMT) by altering the expression of E-cadherin, N-cadherin, and vimentin and induced DNA damage responses. Integrating quantitative proteomics, FHL1 was identified as a potential regulator to modulate EMT after tabersonine treatment. Increased expression of FHL1 was induced by tabersonine treatment, while downregulation of FHL1 reversed the inhibitory effects of tabersonine on OVC cells by mediating EMT. In vivo findings further reflected that the combined treatment of tabersonine and olaparib significantly inhibited tumor growth and OVC metastasis through upregulation of FHL1. Our findings reveal the role of tabersonine in improving the sensitivity of olaparib in OVC through FHL1-mediated EMT, suggesting that tabersonine holds promise for future application in OVC treatment.

Myocardial fibrosis following acute myocardial infarction (AMI) seriously affects the prognosis and survival rate of patients. This study explores the role and regulation mechanism of storax, a commonly used traditional Chinese medicine for treatment of cardiovascular diseases, on myocardial fibrosis and cardiac function. The AMI rat model was established by subcutaneous injection of Isoproterenol hydrochloride (ISO). Storax (0.1, 0.2, 0.4 g/kg) was administered by gavage once/d for 7 days. Electrocardiogram, echocardiography, hemodynamic and cardiac enzyme in AMI rats were measured. HE, Masson, immunofluorescence and TUNEL staining were used to observe the degree of pathological damage, fibrosis and cardiomyocyte apoptosis in myocardial tissue, respectively. Expression of AT1R, CARP and their downstream related apoptotic proteins were detected by WB. The results demonstrated that storax could significantly improve cardiac electrophysiology and function, decrease serum cardiac enzyme activity, reduce type I and III collagen contents to improve fibrosis and alleviate myocardial pathological damage and cardiomyocyte apoptosis. It also found that storax can significantly down-regulate expression of AT1R, Ankrd1, P53, P-p53 (ser 15), Bax and cleaved Caspase-3 and up-regulate expression of Mdm2 and Bcl-2. Taken together, these findings indicated that storax effectively protected cardiomyocytes against myocardial fibrosis and cardiac dysfunction by inhibiting the AT1R-Ankrd1-P53 signaling pathway.

Cachexia, a condition prevalent in many chronically ill patients, is characterized by weight loss, fatigue, and decreases in muscle mass and function. Cachexia is associated with tumor burden and disease-related malnutrition, but other studies implicate chemotherapy as being causative. We investigated the effects of a chemotherapy drug cocktail on myofibrillar protein abundance and synthesis, anabolic signaling mechanisms, and substrate availability. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4 μl/ml DMSO) or a chemotherapy drug cocktail (a mixture of cisplatin [20 μg/ml], leucovorin [10 μg/ml], and 5-fluorouracil [5-FLU; 50 μg/ml]) for 24-72 h. Compared to myotubes treated with vehicle, those treated with the drug cocktail showed 50%-80% reductions in the abundance of myofibrillar proteins, including myosin heavy chain-1, troponin, and tropomyosin (p < 0.05). Cells treated with only a mixture of cisplatin and 5-FLU had identical reductions in myofibrillar protein abundance. Myotubes treated with the drug cocktail also showed >50% reductions in the phosphorylation of AKT Ser473 and of mTORC1 substrates ribosomal protein S6 Ser235/236 , its kinase S6K1 Thr389 and eukaryotic translation initiation factor 4E-binding protein 1 (all p < 0.05). Drug treatment impaired peptide chain initiation in myofibrillar protein fractions and insulin-stimulated glucose uptake (p = 0.06) but increased the expression of autophagy markers beclin-1 and microtubule-associated proteins 1A/1B light chain 3B (p < 0.05), and of apoptotic marker, cleaved caspase 3 (p < 0.05). Drug treatment reduced the expression of mitochondrial markers cytochrome oxidase and succinate dehydrogenase (p < 0.05). The observed profound negative effects of this chemotherapy drug cocktail on myotubes underlie a need for approaches that can reduce the negative effects of these drugs on muscle metabolism., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Context: The early events regulating the remodeling program following skeletal muscle damage are poorly understood., Objective: The objective of this study was to determine the association between myofibrillar protein synthesis (myoPS) and nuclear factor-kappa B (NF-κB) signaling by nutritionally accelerating the recovery of muscle function following damage., Design, Setting, Participants, and Interventions: Healthy males and females consumed daily postexercise and prebed protein-polyphenol (PP; n = 9; 4 females) or isocaloric maltodextrin placebo (PLA; n = 9; 3 females) drinks (parallel design) 6 days before and 3 days after 300 unilateral eccentric contractions of the quadriceps during complete dietary control., Main Outcome Measures: Muscle function was assessed daily, and skeletal muscle biopsies were taken after 24, 27, and 36 hours for measurements of myoPS rates using deuterated water, and gene ontology and NF-κB signaling analysis using a quantitative reverse transcription PCR (RT-qPCR) gene array., Results: Eccentric contractions impaired muscle function for 48 hours in PLA intervention, but just for 24 hours in PP intervention (P = 0.047). Eccentric quadricep contractions increased myoPS compared with the control leg during postexercise (24-27 hours; 0.14 ± 0.01 vs 0.11 ± 0.01%·h-1, respectively; P = 0.075) and overnight periods (27-36 hours; 0.10 ± 0.01 vs 0.07 ± 0.01%·h-1, respectively; P = 0.020), but was not further increased by PP drinks (P > 0.05). Protein-polyphenol drinks decreased postexercise and overnight muscle IL1R1 (PLA = 2.8 ± 0.4, PP = 1.1 ± 0.4 and PLA = 1.9 ± 0.4, PP = 0.3 ± 0.4 log2 fold-change, respectively) and IL1RL1 (PLA = 4.9 ± 0.7, PP = 1.6 ± 0.8 and PLA = 3.7 ± 0.6, PP = 0.7 ± 0.7 log2 fold-change, respectively) messenger RNA expression (P < 0.05) and downstream NF-κB signaling compared with PLA., Conclusion: Protein-polyphenol drink ingestion likely accelerates recovery of muscle function by attenuating inflammatory NF-κB transcriptional signaling, possibly to reduce aberrant tissue degradation rather than increase myoPS rates., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Branched-chain amino acids (BCAA) are one of the most popular sports supplements, marketed under the premise that they enhance muscular adaptations. Despite their prevalent consumption among athletes and the general public, the efficacy of BCAA has been an ongoing source of controversy in the sports nutrition field. Early support for BCAA supplementation was derived from extrapolation of mechanistic data on their role in muscle protein metabolism. Of the three BCAA, leucine has received the most attention because of its ability to stimulate the initial acute anabolic response. However, a substantial body of both acute and longitudinal research has now accumulated on the topic, affording the ability to scrutinize the effects of BCAA and leucine from a practical standpoint. This article aims to critically review the current literature and draw evidence-based conclusions about the putative benefits of BCAA or leucine supplementation on muscle strength and hypertrophy as well as illuminate gaps in the literature that warrant future study.

Herein, we report the effect of flavonoids from Lycium barbarum leaves (LBLF) on myofibrillar proteins (MP) in minced mutton during chilled storage (4 ± 1 ℃). High performance liquid chromatography (HPLC) analysis showed that the total flavonoid content in LBLF was 322.0 mg/g, of which the rutin content was 297.6 mg/g. The effect of 0.5%, 1.0%, and 1.5% LBLF on the structure and thermodynamic properties of MP in minced mutton was studied systematically. Tyrosine and tryptophan of MP samples treated with LBLF were converted from an exposed state to an embedded state. The interaction between LBLF and MP quenched the internal fluorescence, and improved the thermal stability of MP. The addition of LBLF significantly reduced the carbonyl and sulfhydryl contents of MP (p < 0.05), and decreased the surface hydrophobicity of MP in a dose-dependent manner. Our results indicate that LBLF can combine with free radicals produced by protein oxidation, block the free radical oxidation chain reaction, and inhibit the oxidation of MP. Therefore, LBLF may have great potential as a natural antioxidant in meats and meat products during chilled storage. PRACTICAL APPLICATION: Lycium barbarum is widely distributed in China, especially in Qinghai and Ningxia. The results of this study suggest that flavonoids extracted from L. barbarum leaves may be an effective natural antioxidant for the preservation of meats and meat products., (© 2021 Institute of Food Technologists®.)

Lactobacillus-fermented milk can stimulate anabolic effects in skeletal muscle. Fermented milk containing Lactobacillus produces aqueous molecules, such as free AA and lactate. This study aimed to investigate how processing fermented milk by centrifugation and removal of supernatant affects AA absorption and postprandial skeletal muscle protein synthesis (MPS) when mice are fed fermented milk. We gavaged male Sprague-Dawley rats with skim milk (S), fermented milk (F), or processed fermented milk (P), and examined the total AA content in portal vein blood (reflecting AA absorption) and plantaris muscle MPS at 30, 60, and 90 min following administration. Relative to fasted rats, at 30 min the total AA concentration in portal vein blood from rats in the P groups was significantly higher, followed by F and S, respectively. The MPS rates were higher for the F or P groups compared with the S group. Phosphorylation levels of p70S6 kinase in the P and F groups were significantly higher than those for the S group 30 min after administration, although the level of Akt phosphorylation was similar among the groups. These results suggested that fermentation improves AA absorption that in turn enhances postprandial MPS via Akt-independent mechanisms, and that processed fermented milk retains these favorable effects on MPS., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Hypobaric hypoxic stress leads to oxidative stress, inflammation, and disturbance in protein turnover rate. Aggregately, this imbalance in redox homeostasis is responsible for skeletal muscle protein loss and a decline in physical performance. Hence, an urgent medical need is required to ameliorate skeletal muscle protein loss. The present study investigated the efficacy of ursolic acid (UA), a pentacyclic triterpene acid to ameliorate hypobaric hypoxia (HH)-induced muscle protein loss. UA is a naturally occurring pentacyclic triterpene acid present in several edible herbs and fruits such as apples. It contains skeletal muscle hypertrophy activity; still its potential against HH-induced muscle protein loss is unexplored. To address this issue, an in vivo study was planned to examine the beneficial effect of UA supplementation on HH-induced skeletal muscle loss. Male Sprague Dawley rats were exposed to HH with and without UA supplementation (20 mg/kg; oral) for 3 continuous days. The results described the beneficial role of UA as supplementation of UA with HH exposure attenuated reactive oxygen species production and oxidative protein damage, which indicate the potent antioxidant activity. Furthermore, UA supplementation enhanced Akt, pAkt, and p70S6kinase activity (Akt pathway) and lowered the pro-inflammatory cytokines in HH exposed rats. UA has potent antioxidant and anti-inflammatory activity, and it enhanced the protein content via upregulation of Akt pathway-related proteins against HH exposure. These three biological activities of UA make it a novel candidate for amelioration of HH-induced skeletal muscle damage and protein loss., (© 2020 International Union of Biochemistry and Molecular Biology.)

Isoleucine (Ile), as a branched-chain amino acid (BCAA), has a vital role in regulating body weight and muscle protein synthesis. However, the regulatory effect of Ile on muscle mass under high-fat diet (HFD) conditions and intramyocellular lipid deposition remains largely unclear. In this study, a feeding experiment with HFD with or without 25 g L -1 Ile was performed using 32 wild male C57BL/6J mice randomly divided into two groups. The results showed that Ile significantly increased both muscle and fat mass, as well as causing insulin resistance and meanwhile upregulating the levels of key adipogenic and myogenic proteins. More importantly, Ile damaged the mitochondrial function by vacuolation, swelling and cristae fracture in the gastrocnemius (GAS) and tibialis anterior (TA) with downregulation of mitochondrial function-related genes. Furthermore, Ile promoted myogenesis and more lipid droplet accumulation in myotubes. Compared with the control, the protein levels of myosin heavy chain (MyHC), myoblast determination protein 1 (MyoD), myogenin (MyoG), peroxisome proliferator-activated receptor gamma (PPARg) and fatty acid synthase (FAS) were upregulated in the Ile group, whereas the protein levels of adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL) were downregulated. Collectively, Ile increased muscle mass through myogenesis and intramyocellular lipid deposition. Our findings provide a new perspective for not only improving the lean juiciness of farm animals by increasing intramyocellular lipid accumulation, but also modulating myopathies under obesity.

Background: Fortetropin is a proteo-lipid complex made from fertilized egg yolk and, in young men, has been shown to increase lean body mass., Methods: The purpose of this study was to examine the effects of 21 days of Fortetropin supplementation on the fractional synthetic rate (FSR) of muscle protein in 10 healthy, older men and 10 women (66.4 ± 4.5 y). We used 2H2O labeling to measure FSR of multiple muscle protein ontologies. D3-creatine dilution was used to determine muscle mass at baseline. Subjects ingested 70% 2H2O for 21 day and saliva samples were collected to determine body 2H2O enrichment. A microbiopsy was obtained from the m. vastus lateralis on Day 21. Subjects were randomly assigned to Fortetropin (19.8 g/d) or placebo (cheese powder, 19.8 g/d)., Results: Restricting kinetic data to proteins with ≥2 peptides measured in at least 4 subjects per group resulted in 117 proteins meeting these criteria. The mean FSR for a majority of proteins in several muscle gene ontologies was higher in the Fortetropin group compared to placebo (32/38 myofibril proteins, 33/44 sarcoplasmic proteins, and 12/17 mitochondrial proteins) and this proportion was significantly different between groups using a binomial test and were independent of sex or baseline muscle mass., Conclusions: The overall magnitude of the difference in muscle protein FSR of Fortetropin from placebo was 18%, with multiple gene ontologies affected. While these results should be confirmed in larger cohorts, they suggest that Fortetropin supplementation is effective for promoting muscle protein synthesis in older people., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Gerontological Society of America.)

Protein turnover is a process that is regulated by several factors and can lead to muscle hypertrophy or atrophy. The purpose of the present study was to determine the effects of β-hydroxy-β-methylbutyrate free acid (HMB-FA) and eccentric resistance exercise on variables related to protein turnover in rats. Thirty-two male rats were randomly assigned into four groups of eight, including control, control-HMB, exercise, and exercise-HMB. Animals in HMB groups received 340 mg/kg/day for two weeks. Animals in the exercise groups performed one session of eccentric resistance exercise consisting of eight repetitions descending from a ladder with a slope of 80 degree, with an extra load of two times body weight (100% 1RM). Twenty-four hours after the exercise session, triceps brachii muscle and serum were collected for further analysis. Exercise and HMB-FA induced lower muscle myostatin and higher muscle Fibronectin type III domain containing 5 (FNDC5), P70-S6 kinase 1 gene expression, as well as higher serum irisin and IGF-1 concentrations. Exercise alone induced higher caspase-3 and caspase-8 gene expression while HMB-FA alone induced lower caspase 3 gene expression. HMB-FA supplement increased the effect of exercise on muscle FNDC5, myostatin, and P70-S6 kinase 1 gene expression. The interaction of exercise and HMBFA resulted in an additive effect, increasing serum irisin and IGF-1 concentrations. In conclusion, a 2-week HMB-FA supplementation paired with acute eccentric resistance exercise can positively affect some genes related to muscle protein turnover., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Aberrant activation of epidermal growth factor receptor (EGFR) signaling pathway is associated with the pathogenesis of pulmonary hypertension (PH). However, the effect of icotinib, a first generation of EGFR tyrosine kinase inhibitor (EGFR-TKI), on PH remains to be elucidated., Methods: PH rat model was established by a single intraperitoneal injection of monocrotaline (MCT, 60 mg/kg). Icotinib (15, 30, and 60 mg/kg/day) was administered by oral gavage from the day of MCT injection. After 4 weeks, hemodynamic parameters and histological changes of the pulmonary arterial vessels were assessed, and the phenotypic switching of pulmonary arterial smooth muscle cells (PASMCs) was determined in vivo. Moreover, the effects of icotinib (10 µM) on epidermal growth factor (EGF, 50 ng/ml)-stimulated proliferation, migration, and phenotypic switching of human PASMCs were explored in vitro., Results: Icotinib significantly reduced the right ventricular systolic pressure and right ventricle hypertrophy index in rats with MCT-induced PH. Moreover, icotinib improved MCT-induced pulmonary vascular remodeling. The expression of contractile marker (smooth muscle 22 alpha (SM22α)) and synthetic markers (osteopontin (OPN) and vimentin) in pulmonary artery was restored by icotinib treatment. In vitro, icotinib suppressed EGF-induced PASMCs proliferation and migration. Meanwhile, icotinib inhibited EGF-induced downregulation of α-smooth muscle actin and SM22α and upregulation of OPN and Collagen I in PASMCs, suggesting that icotinib could inhibit EGF-induced phenotypic switching of PASMCs. Mechanistically, these effects of icotinib were associated with the inhibition of EGFR-Akt/ERK signaling pathway., Conclusions: Icotinib can attenuate MCT-induced pulmonary vascular remodeling and improve PH. This effect of icotinib might be attributed to preventing PASMC dysfunction by inhibiting EGFR-Akt/ERK signaling pathway., (© American Journal of Hypertension, Ltd 2020. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: We have previously evaluated muscle functions and morphology in power athletes of long term (5 to15 years) abuse of anabolic androgen steroids (AAS; Doped) and in clean power athletes (Clean), and observed significant improvements in both muscle morphology and muscle functions in Doped. To our knowledge, the effects of long term AAS abuse on human muscle protein profile have never been studied., Methods: The study examined further the muscle biopsies using a two-dimensional difference gel electrophoresis (2D DIGE) for proteomic screening and protein expression. Cellular localization/distribution of specific proteins identified by proteomic analysis was examined using immunohistochemistry (IHC)., Results: Different protein profiles were observed between Doped and Clean, and a valid orthogonal projection of latent structure discriminant analysis model was built (N.=16, x=5, R2=0.88/Q2=0.84, P=0.0005), which separated Doped from Clean. Liquid chromatography followed by tandem spectrometry identified 14 protein spots (representing nine different proteins) of significant difference in relative quantity (P<0.05), of which nine spots were down-regulated in Doped compared with Clean. IHC revealed no significant alteration in cellular localization in phosphoglucomutase-1 and heat shock protein beta-1, but indeed in two reference proteins desmin and F-actin in Doped., Conclusions: Long term abuse of AAS in combination with training is potentially associated with alterations in skeletal muscle protein profile and protein expression, and structural proteins rather than non-structural proteins are preferentially affected in cellular localization/distribution.

Recently, great attention has been directed towards the use of essential oils from aromatic plants as antimicrobials and antioxidant in food matrix. Fish is well known to be a high perishable food. Indeed, fish muscle is susceptible to suffer protein and lipid oxidation during frozen storage, which can lead to the development of softening and undesirable volatile molecules. However, the possible inclusion of essential oils in fish feed for preserving fish flesh quality during storage is still unclear. For this reason, the potential protective effects of the incorporation of a dietary essential oil constituted by eucalyptol, carvacrol and thymol, to rainbow trout's (Oncorhynchus mykiss) feed were here investigated. Frozen fish fillets resulting from trout fed the essential oil showed a significant protection of specific muscle proteins against the oxidation produced during frozen storage at -10ºC for 6 months. Essential oil-enriched feed decreased carbonylation of specific myofibrillar (α-actinins-1 and -3, myosin heavy chain, myomesin-1, pyruvate kinase, tropomyosin, troponin-T and actin) and sarcoplasmic proteins (glycogen phosphorylase, creatine kinase, fructose-bisphosphate aldolase A and phosphoglycerate mutase 2). Essential oils also increased actin stability and preserved muscle protein solubility and water holding capacity. In addition, essential oils inhibited the onset of lipid oxidation and rancidity, resulting in frozen fish with superior textural quality and sensory scores. As a final conclusion, the inclusion of essential oils in farmed rainbow trout feed is largely efficient for increasing fish quality and shelf life during frozen storage, mainly through a selective-antioxidant effect on muscle proteins.

Introduction: We recently demonstrated the beneficial effects of 4-aminopyridine (4-AP), a potassium channel blocker, in enhancing remyelination and recovery of nerve conduction velocity and motor function after sciatic nerve crush injury in mice. Although muscle atrophy occurs very rapidly after nerve injury, the effect of 4-AP on muscle atrophy and intrinsic muscle contractile function is largely unknown., Methods: Mice were assigned to sciatic nerve crush injury and no-injury groups and were followed for 3, 7, and 14 days with/without 4-AP or saline treatment. Morphological, functional, and transcriptional properties of skeletal muscle were assessed., Results: In addition to improving in vivo function, 4-AP significantly reduced muscle atrophy with increased muscle fiber diameter and contractile force. Reduced muscle atrophy was associated with attenuated expression of atrophy-related genes and increased expression of proliferating stem cells., Discussion: These findings provide new insights into the potential therapeutic benefits of 4-AP against nerve injury-induced muscle atrophy and dysfunction. Muscle Nerve 60: 192-201, 2019., (© 2019 Wiley Periodicals, Inc.)

Background: Age-related muscle loss (sarcopenia) may be driven by a diminished myofibrillar protein synthesis (MyoPS) response to anabolic stimuli (i.e. exercise and nutrition). Oral phosphatidic acid (PA) ingestion has been reported to stimulate resting muscle protein synthesis in rodents, and enhance resistance training-induced muscle remodelling in young humans., Purpose: This study examined the effects of acute oral PA ingestion on resting and exercise-induced MyoPS rates in older individuals., Methods: Sixteen older males performed a bout of unilateral leg resistance exercise followed by oral ingestion of 750 mg of soy-derived PA or a rice-flour placebo (PL) over 60 min post-exercise. A primed-continuous infusion of l-[ring- 13 C 6 ]-phenylalanine with serial muscle biopsies was used to determine MyoPS at rest and between 0-150 and 150-300 min post-exercise., Results: Plasma [PA] concentrations were elevated above basal values from 180 to 300 min post-exercise in PA only (P = 0.02). Exercise increased MyoPS rates above basal values between 150 and 300 min post-exercise in PL (P = 0.001), but not PA (P = 0.83). Phosphorylation of p70S6K, rpS6, 4E-BP1 and Akt was elevated above basal levels in the exercised leg over 150-300 min post-exercise for PL only (P = 0.018, 0.007, 0.011 and 0.002, respectively), and were significantly greater than PA (P < 0.01 for all proteins). The effects of oral PA ingestion on proteolytic signaling markers were equivocal., Conclusions: Acute oral phosphatidic acid ingestion appears to interfere with resistance exercise-induced intramuscular anabolic signaling and MyoPS in older males and, therefore, may not be a viable treatment to counteract sarcopenia. Clinicaltials.gov registration no: NCT03446924., (Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPβ gene. Notably, C/EBPβ also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPβ and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPβ alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPβ binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPβ. C/EBPβ binds directly to the promoter of the musclin gene and upregulates its expression.

Background: Citrulline (CIT), is not extracted by the splanchnic area, can stimulate muscle protein synthesis and could potentially find clinical applications in conditions involving low amino acid (AA) intake, such as in malnourished older subjects., Objective: Our purpose was to research the effects of CIT supplementation on protein metabolism in particular on non-oxidative leucine disposal (NOLD, primary endpoint), and splanchnic extraction of amino acids in malnourished older patients., Design: This prospective randomized multicenter study determined whole-body and liver protein synthesis, splanchnic protein metabolism and appendicular skeletal muscle mass (ASMM) in 24 malnourished older patients [80-92 years; 18 women and 6 men] in inpatient rehabilitation units. All received an oral dose of 10 g of CIT or an equimolar mixture of six non-essential amino acids (NEAAs), as isonitrogenous placebo, for 3 weeks., Results: NOLD and albumin fractional synthesis rates were not different between the NEAA and CIT groups. Splanchnic extraction of dietary amino acid tended to decrease (p = 0.09) in the CIT group (45.2%) compared with the NEAA group (60.3%). Total differences in AA and NEAA area under the curves between fed-state and postabsorptive-state were significantly higher in the CIT than in the NEAA group. There were no significant differences for body mass index, fat mass (FM), lean mass (LM) or ASMM in the whole population except for a tendential decrease in FM for the citrulline group (p = 0.089). Compared with Day 1, lean mass and ASMM significantly increased (respectively p = 0.016 and p = 0.018) at Day 20 in CIT-treated women (mean respective increase of 1.7 kg and 1.1 kg), and fat mass significantly decreased (p = 0.001) at Day 20 in CIT-group women (mean decrease of 1.3 kg)., Conclusions: Our results demonstrate that CIT supplementation has no effect on whole-body protein synthesis or liver protein synthesis in malnourished older subjects. However, CIT supplementation was associated with a higher systemic AA availability. In the subgroup of women, CIT supplementation increased LM and ASMM, and decreased FM., (Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

BACKGROUND The inhibitory effect of arsenic trioxide (As₂O₃) on lung cancer has been reported in some preclinical studies. However, its effect on small cell lung cancer (SCLC) has been poorly explored. Calcineurin and its substrate, nuclear factor of activated T cells (NFAT), mediate the downstream signaling of VEGF, and is critical in the process endothelium activation and tumor metastasis. In this study, we aimed to evaluate whether As₂O₃ had inhibitory effects on endothelial cells activation and the metastasis of SCLC, and to explore the possible mechanisms. MATERIAL AND METHODS In vitro, human umbilical vein endothelial cells (HUVECs) were used. Cell Counting Kit-8 assay and cell migration assay were performed to determine the effect of As₂O₃ on HUVECs proliferation and migration. The level of calcineurin, NFAT, downstream factors for Down syndrome candidate region 1 (DSCR1), and the endogenous inhibitor of calcineurin, were evaluated by quantitative PCR and western blotting. In vivo, SCLC metastasis models were established by injecting NCI-H446 cells into tail veins of nude mice. Tumor-bearing mice were treated with As₂O₃ or calcineurin inhibitor for 10 days, after which tumor metastasis in target organs was evaluated. RESULTS As₂O₃ significantly inhibited the proliferation and migration of endothelial cells. Also, As₂O₃ inhibited the expression levels of calcineurin, NFAT, and the downstream target genes CXCR7 and RND1, while it upregulated the level of DSCR1. Both As₂O₃ and calcineurin inhibitor exhibited notable inhibitory effect on the metastasis of SCLC, without obvious side effects. CONCLUSIONS These findings suggested that As₂O₃ had remarkable inhibitory effects on the endothelial cell activation and SCLC metastasis, and the mechanism might be related to the blocking of calcineurin-NFAT signaling by upregulating DSCR1.

Objective: S1P has known endothelial barrier-protective properties, but whether this extends to the BBB is unclear. We hypothesized that alcohol-induced disruption of brain microvascular endothelial barrier function and junctional protein organization can be ameliorated by S1P treatment., Methods: Cultured primary HBMEC monolayers were used to characterize endothelial-specific mechanisms of BBB regulation. TER and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization of Claudin-5, VE-cadherin, and β-catenin was determined by immunofluorescence confocal microscopy. S1P was applied following treatment with alcohol., Results: Alcohol significantly impaired HBMEC TER. Application of S1P after alcohol treatment resulted in a hastened recovery to the baseline HBMEC TER. Alcohol-treated HBMEC had a significantly higher mean permeability than control that was reversed by S1P. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization of VE-Cadherin, Claudin-5, and β-catenin., Conclusions: Alcohol impairs the barrier function and junctional organization of HBMEC monolayers. S1P enhanced barrier function and restored junctions in the presence of alcohol, and thus may be useful for restoring BBB function during alcohol intoxication., (© 2018 John Wiley & Sons Ltd.)

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.

Background: Acute inflammation, and subsequent release of bacterial products (e.g. LPS), inflammatory cytokines, and stress hormones, is catabolic, and the loss of lean body mass predicts morbidity and mortality. Lipid intermediates may reduce protein loss, but the roles of free fatty acids (FFAs) and ketone bodies during acute inflammation are unclear., Objective: We aimed to test whether infusions of 3-hydroxybutyrate (3OHB), FFAs, and saline reduce protein catabolism during exposure to LPS and Acipimox (to restrict and control endogenous lipolysis)., Design: A total of 10 healthy male subjects were randomly tested 3 times, with: 1) LPS, Acipimox (Olbetam) and saline, 2) LPS, Acipimox, and nonesterified fatty acids (Intralipid), and 3) LPS, Acipimox, and 3OHB, during a 5-h basal period and a 2-h hyperinsulinemic, euglycemic clamp. Labeled phenylalanine, tyrosine, and urea tracers were used to estimate protein kinetics, and muscle biopsies were taken for Western blot analysis of protein metabolic signaling., Results: 3OHB infusion increased 3OHB concentrations (P < 0.0005) to 3.5 mM and decreased whole-body phenylalanine-to-tyrosine degradation. Basal and insulin-stimulated net forearm phenylalanine release decreased by >70% (P < 0.005), with both appearance and phenylalanine disappearance being profoundly decreased. Phosphorylation of eukaryotic initiation factor 2α at Ser51 was increased in skeletal muscle, and S6 kinase phosphorylation at Ser235/236 tended (P = 0.074) to be decreased with 3OHB infusion (suggesting inhibition of protein synthesis), whereas no detectable effects were seen on markers of protein breakdown. Lipid infusion did not affect phenylalanine kinetics, and insulin sensitivity was unaffected by interventions., Conclusion: During acute inflammation, 3OHB has potent anticatabolic actions in muscle and at the whole-body level; in muscle, reduction of protein breakdown overrides inhibition of synthesis. This trial was registered at clinicaltrials.gov as NCT01752348.

Human myofibrillogenesis regulator 1 (MR-1) is a functional gene also known as paroxysmal nonkinesigenic dyskinesia (PNKD). It is localised on human chromosome 2q35 and three different isomers, MR-1L, MR-1M and MR-1S, are formed by alternative splicing. MR-1S promotes cardiac hypertrophy and is closely related to cancer. MR-1S is overexpressed in haematologic and solid malignancies, such as hepatoma, breast cancer and chronic myelogenous leukaemia. MR-1S causes disordered cell differentiation, initiates malignant transformation and accelerates metastasis. MR-1S directly phosphorylates and activates the MEK-ERK-RSK pathway to accelerate cancer growth and facilitates metastasis by activating the MLC2-FAK-AKT pathway. Silencing MR-1 inhibits cancer cell proliferation and metastasis. MR-1S causes disordered cell differentiation, initiates malignant transformation and accelerates metastasis. MR-1 interacts with eukaryotic translation initiation factors and MRIP-1, which contains Ras GTPase, PH and zinc-containing ArfGap domains, as well as three ankyrin repeats. Mutations in the N-terminal region of MR-1L and MR-1S are the main causes of PNKD (a hereditary disease characterised by paroxysmal dystonic choreoathetosis) and targeting the mutated protein could provide symptomatic relief. These findings provide compelling evidence that MR-1 might be a diagnostic marker and therapeutic target for solid tumours, myelogenous leukaemia and PNKD.

Introduction: Muscle wasting is a frequent, debilitating complication of cancer. The impact of colorectal cancer chemotherapeutic oxaliplatin on the development of muscle loss and associated molecular changes is of clinical importance., Methods: C57BL/6J male mice were treated with oxaliplatin. Total body weights were measured and behavioral studies performed. Hindlimb muscle weights (gastrocnemius and soleus) were recorded in conjunction with gene and protein expression analysis., Results: Oxaliplatin-treated mice displayed reduced weight gain and behavioral deficits. Mice treated over a shorter course had significantly increased STAT3 phosphorylation in gastrocnemius muscles. Mice receiving extended oxaliplatin treatment demonstrated reduced hindlimb muscle mass with upregulation of myopathy-associated genes Foxo3, MAFbx, and Bnip3., Discussion: The findings suggest that oxaliplatin treatment can directly disrupt skeletal muscle homeostasis and promote muscle loss, which may be clinically relevant in the context of targeting fatigue and weakness in cancer patients. Muscle Nerve 57: 650-658, 2018., (© 2017 Wiley Periodicals, Inc.)

Background: Different techniques have been applied to alter myofibril protein (MP) structure, which further promotes protein-protein interactions and influencing the MP gelling characteristics. Influence of BslA from natto food (protein concentration, 30 mg mL -1 ; at 0.001, 0.005, 0.01, 0.05 and 0.1 g kg -1 ) on the characteristics of MP gel of chicken breast was investigated., Results: Results show that cooking loss significantly (P < 0.05) decreased with increased percentage of BslA. Hardness of MP gel did not significantly change at 0.01 g kg -1 BslA. Differential scanning calorimetry disclosed that MP was modified by the addition of BslA. Moreover, BslA produced a high value of storage modulus (G') and low value of phase angle (tan δ) during heating, especially at 0.01 g kg -1 . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis proved the formation of higher-molecular-weight polymers by developing non-disulfide covalent bonds between MP at 0.01 g kg -1 BslA. Surface hydrophobicity of the MP gel was decreased with increased percentage of BslA. Scanning electron microscopy confirmed the increasing number of uniform cavities of MP gel with the increased percentage of BslA., Conclusion: Addition of 0.01 g kg -1 BslA significantly improved the water holding capacity and rheological properties of MP by developing non-disulfide covalent bonds. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

Sarcopenia or skeletal muscle loss is a frequent, potentially reversible complication in cirrhosis that adversely affects clinical outcomes. Hyperammonemia is a consistent abnormality in cirrhosis that results in impaired skeletal muscle protein synthesis and breakdown (proteostasis). Despite the availability of effective ammonia-lowering therapies, whether lowering ammonia restores proteostasis and increases muscle mass is unknown. Myotube diameter, protein synthesis, and molecular responses in C2C12 murine myotubes to withdrawal of ammonium acetate following 24-hour exposure to 10 mM ammonium acetate were complemented by in vivo studies in the hyperammonemic portacaval anastomosis rat and sham-operated, pair-fed Sprague-Dawley rats treated with ammonia-lowering therapy by l-ornithine l-aspartate and rifaximin orally for 4 weeks. We observed reduced myotube diameter, impaired protein synthesis, and increased autophagy flux in response to hyperammonemia, which were partially reversed following 24-hour and 48-hour withdrawal of ammonium acetate. Consistently, 4 weeks of ammonia-lowering therapy resulted in significant lowering of blood and skeletal muscle ammonia, increase in lean body mass, improved grip strength, higher skeletal muscle mass and diameter, and an increase in type 2 fibers in treated compared to untreated portacaval anastomosis rats. The increased skeletal muscle myostatin expression, reduced mammalian target of rapamycin complex 1 function, and hyperammonemic stress response including autophagy markers normally found in portacaval anastomosis rats were reversed by treatment with ammonia-lowering therapy. Despite significant improvement, molecular and functional readouts were not completely reversed by ammonia-lowering measures., Conclusion: Ammonia-lowering therapy results in improvement in skeletal muscle phenotype and function and molecular perturbations of hyperammonemia; these preclinical studies complement previous studies on ammonia-induced skeletal muscle loss and lay the foundation for prolonged ammonia-lowering therapy to reverse sarcopenia of cirrhosis. (Hepatology 2017;65:2045-2058)., (© 2017 by the American Association for the Study of Liver Diseases.)

Maximizing anabolic responses to feeding and exercise is crucial for muscle maintenance and adaptation to exercise training. We hypothesized that enriching a protein drink with leucine would improve anabolic responses to resistance exercise (RE: 6 × 8 knee-extension repetitions at 75% of 1-RM) in both young and older adults. Groups (n = 9) of young (24 ± 6 y, BMI 23 ± 2 kg m -2 ) and older men (70 ± 5 y, BMI 25 ± 2 kg m -2 ) were randomized to either: (i) RE followed by Slim-Fast Optima (SFO 10 g PRO; 24 g CHO) with 4.2 g of leucine (LEU) or, (ii) RE + SFO with 4.2 g of alanine (ALA; isonitrogenous control). Muscle biopsies were taken before, immediately after, and 1, 2 and 4 h after RE and feeding. Muscle protein synthesis (MPS) was measured by incorporation of [1, 2- 13 C 2 ] leucine into myofibrillar proteins and the phosphorylation of p70S6K1 by immunoblotting. In young men, both area under the curve (AUC; FSR 0-4 h P < 0.05) and peak FSR (0.11 vs. 0.08%.h. -1 ; P < 0.05) were greater in the SFO + LEU than in the SFO + ALA group, after RE. Similarly, in older men, AUC analysis revealed that post-exercise anabolic responses were greater in the SFO + LEU than SFO + ALA group, after RE (AUC; FSR 0-4 h P < 0.05). Irrespective of age, increases in p70S6K1 phosphorylation were evident in response to both SFO + LEU and SFO + ALA, although greater with leucine supplementation than alanine (fold-change 2.2 vs. 3.2; P < 0.05), specifically in the older men. We conclude that addition of Leucine to a sub-maximal PRO bolus improves anabolic responses to RE in young and older men., (Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1-10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our analysis revealed that caffeine promoted a 40% increase in autolysosome formation and a 25% increase in autophagic flux. In contrast, caffeine treatment did not significantly increase the expression of the skeletal muscle specific ubiquitin ligases MAFbx and MuRF1 or 20S proteasome activity. Caffeine treatment significantly reduced mTORC1 signaling, total protein synthesis and myotube diameter in a CaMKKβ/AMPK-dependent manner. Further, caffeine promoted a CaMKII-dependent increase in myostatin mRNA expression that did not significantly contribute to the caffeine-dependent reduction in protein synthesis. Our results indicate that short-term caffeine exposure significantly reduced skeletal myotube diameter by increasing autophagic flux and promoting a CaMKKβ/AMPK-dependent reduction in protein synthesis.

Context: Androgen deprivation therapy (ADT) is a common prostate cancer (PCa) treatment but results in muscular atrophy. Periodic increases in muscle protein synthesis (MPS) that occur after resistance exercise or protein intake may ameliorate this muscle loss, but the impact of these anabolic stimuli during ADT is unclear., Objective: To determine the acute MPS response to whey protein supplementation with and without resistance exercise during ADT., Design: Acute response in PCa patients vs age-matched controls (CON)., Setting: Academic laboratory setting., Participants: PCa patients on ADT (N = 8) and CON (N = 10)., Intervention: A standardized diet was consumed for 2 days prior to performing unilateral knee extension resistance exercise followed by ingestion of 40 g of whey protein., Main Outcome Measures: Bilateral biopsies and stable isotope infusions were used to determine MPS rates at rest after protein ingestion with and without resistance exercise., Results: Baseline MPS during ADT was suppressed relative to CON (P = 0.01). Protein consumption stimulated MPS in both groups (approximate twofold increase, both P < 0.001), but to a greater extent in CON (P = 0.003). Protein plus resistance exercise increased MPS (∼3.4-fold increase, both P < 0.001) to a greater extent than did protein alone (P < 0.001), but with no difference between groups (P = 0.380)., Conclusions: ADT reduces basal and protein feeding-induced rises in MPS; however, combined protein ingestion with resistance exercise stimulated MPS to a similar degree as CON. Testosterone appears to play a role in maintaining muscle mass but is not necessary to initiate a robust response in MPS following resistance exercise when combined with protein ingestion., (Copyright © 2017 by the Endocrine Society)

Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P < 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control., (Copyright © 2017 the American Physiological Society.)

Protein ingestion before sleep augments postexercise muscle protein synthesis during overnight recovery. It is unknown whether postexercise and presleep protein consumption modulates postprandial protein handling and myofibrillar protein synthetic responses the following morning. Sixteen healthy young (24 ± 1 yr) men performed unilateral resistance-type exercise (contralateral leg acting as a resting control) at 2000. Participants ingested 20 g of protein immediately after exercise plus 60 g of protein presleep (PRO group; n = 8) or equivalent boluses of carbohydrate (CON; n = 8). The subsequent morning participants received primed, continuous infusions of l-[ring- 2 H 5 ]phenylalanine and l-[1- 13 C]leucine combined with ingestion of 20 g intrinsically l-[1- 13 C]phenylalanine- and l-[1- 13 C]leucine-labeled protein to assess postprandial protein handling and myofibrillar protein synthesis in the rested and exercised leg in CON and PRO. Exercise increased postabsorptive myofibrillar protein synthesis rates the subsequent day (P < 0.001), with no differences between CON and PRO. Protein ingested in the morning increased myofibrillar protein synthesis in both the exercised and rested leg (P < 0.01), with no differences between treatments. Myofibrillar protein bound l-[1- 13 C]phenylalanine enrichments were greater in the exercised (0.016 ± 0.002 and 0.015 ± 0.002 MPE in CON and PRO, respectively) vs. rested (0.010 ± 0.002 and 0.009 ± 0.002 MPE in CON and PRO, respectively) leg (P < 0.05), with no differences between treatments (P > 0.05). The additive effects of resistance-type exercise and protein ingestion on myofibrillar protein synthesis persist for more than 12 h after exercise and are not modulated by protein consumption during acute postexercise recovery. This work provides evidence of an extended window of opportunity where presleep protein supplementation can be an effective nutrient timing strategy to optimize skeletal muscle reconditioning., (Copyright © 2016 the American Physiological Society.)

Background: Based on circulating C-reactive protein (CRP) levels, some individuals develop slightly increased inflammation as they age. In elderly inflamed rats, the muscle response to protein feeding is impaired, whereas it can be maintained by treatment with non-steroidal anti-inflammatory drugs (NSAIDs). It is unknown whether this applies to elderly humans with increased inflammation. Thus, the muscle response to whey protein bolus ingestion with and without acute resistance exercise was compared between healthy elderly individuals and elderly individuals with slightly increased inflammation±NSAID treatment., Methods: Twenty-four elderly men (>60years) were recruited. Of those, 14 displayed a slightly increased systemic inflammation (CRP>2mg/l) and were randomly assigned to NSAID (Ibuprofen 1800mg/day) or placebo treatment for 1week. The remaining 10 elderly individuals served as healthy controls (CRP<1mg/l). The muscle protein synthetic response was measured as the fractional synthetic rate (FSR) and p70S6K phosphorylation-to-total protein ratio., Results: The basal myofibrillar FSR and the myofibrillar FSR responses to whey protein bolus ingestion with and without acute resistance exercise were maintained in inflamed elderly compared to healthy controls (p>0.05) and so was p70S6K phosphorylation. Moreover, NSAID treatment did not significantly improve the myofibrillar and connective tissue FSR responses or reduce the plasma CRP level in inflamed, elderly individuals (p>0.05)., Conclusion: A slight increase in systemic inflammation does not affect the basal myofibrillar FSR or the myofibrillar FSR responses, which suggests that elderly individuals with slightly increased inflammation can benefit from protein ingestion and resistance exercise to stimulate muscle protein anabolism. Moreover, the NSAID treatment did not significantly affect the myofibrillar or connective tissue FSR responses to protein ingestion and acute resistance exercise., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The age-related anabolic resistance to protein ingestion is suggested to be associated with impairments in insulin-mediated capillary recruitment and postprandial muscle tissue perfusion. The present study investigated whether dietary nitrate co-ingestion with protein improves muscle protein synthesis in older, type 2 diabetes patients. Twenty-four men with type 2 diabetes (72 ± 1 yr, 26.7 ± 1.4 m/kg(2) body mass index, 7.3 ± 0.4% HbA1C) received a primed continuous infusion of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine and ingested 20 g of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled protein with (PRONO3) or without (PRO) sodium nitrate (0.15 mmol/kg). Blood and muscle samples were collected to assess protein digestion and absorption kinetics and postprandial muscle protein synthesis rates. Upon protein ingestion, exogenous phenylalanine appearance rates increased in both groups (P < 0.001), resulting in 55 ± 2% and 53 ± 2% of dietary protein-derived amino acids becoming available in the circulation over the 5h postprandial period in the PRO and PRONO3 groups, respectively. Postprandial myofibrillar protein synthesis rates based on l-[ring-(2)H5]phenylalanine did not differ between groups (0.025 ± 0.004 and 0.021 ± 0.007%/h over 0-2 h and 0.032 ± 0.004 and 0.030 ± 0.003%/h over 2-5 h in PRO and PRONO3, respectively, P = 0.7). No differences in incorporation of dietary protein-derived l-[1-(13)C]phenylalanine into de novo myofibrillar protein were observed at 5 h (0.016 ± 0.002 and 0.014 ± 0.002 mole percent excess in PRO and PRONO3, respectively, P = 0.8). Dietary nitrate co-ingestion with protein does not modulate protein digestion and absorption kinetics, nor does it further increase postprandial muscle protein synthesis rates or the incorporation of dietary protein-derived amino acids into de novo myofibrillar protein in older, type 2 diabetes patients., (Copyright © 2016 the American Physiological Society.)

Introduction: In this study we investigated the effects of high-intensity resistance training (RT) on dexamethasone (DEX)-induced muscle atrophy in flexor hallucis longus (FHL), tibialis anterior (TA), and soleus (SOL) muscles., Methods: Rats underwent either high-intensity RT or were kept sedentary. In the last 10 days they received either DEX (0.5 mg/kg/day, intraperitoneally) or saline., Results: DEX reduced body weight (-21%), food intake (-28%), FHL and TA muscle mass (-20% and -18%, respectively), and increased muscle-specific ring finger 1 (MuRF-1) protein level (+37% and +45.5%). RT attenuated FHL muscle atrophy through a combination of low increase in MuRF-1 protein level (-3.5%) and significant increases in mammalian target of rapamycin (mTOR) (+63%) and p70S6K (+46% and +49% for control and DEX, respectively) protein levels., Conclusion: RT attenuated DEX-induced muscle atrophy through a combination of increases in mTOR and p70S6K protein levels and a low increase in MuRF-1 protein level., (© 2016 Wiley Periodicals, Inc.)

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 μmol·kg -1 ·h -1 ) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM., (Copyright © 2016 the American Physiological Society.)

Diabetic kidney disease (DKD) is a microvascular complication that leads to kidney dysfunction and ESRD, but the underlying mechanisms remain unclear. Podocyte Wnt-pathway activation has been demonstrated to be a trigger mechanism for various proteinuric diseases. Notably, four-and-a-half LIM domains protein 2 (FHL2) is highly expressed in urogenital systems and has been implicated in Wnt/β-catenin signaling. Here, we used in vitro podocyte culture experiments and a streptozotocin-induced DKD model in FHL2 gene-knockout mice to determine the possible role of FHL2 in DKD and to clarify its association with the Wnt pathway. In human and mouse kidney tissues, FHL2 protein was abundantly expressed in podocytes but not in renal tubular cells. Treatment with high glucose or diabetes-related cytokines, including angiotensin II and TGF-β1, activated FHL2 protein and Wnt/β-catenin signaling in cultured podocytes. This activation also upregulated FHL2 expression and promoted FHL2 translocation from cytosol to nucleus. Genetic deletion of the FHL2 gene mitigated the podocyte dedifferentiation caused by activated Wnt/β-catenin signaling under Wnt-On, but not under Wnt-Off, conditions. Diabetic FHL2(+/+) mice developed markedly increased albuminuria and thickening of the glomerular basement membrane compared with nondiabetic FHL2(+/+) mice. However, FHL2 knockout significantly attenuated these DKD-induced changes. Furthermore, kidney samples from patients with diabetes had a higher degree of FHL2 podocyte nuclear translocation, which was positively associated with albuminuria and progressive renal function deterioration. Therefore, we conclude that FHL2 has both structural and functional protein-protein interactions with β-catenin in the podocyte nucleus and that FHL2 protein inhibition can mitigate Wnt/β-catenin-induced podocytopathy., (Copyright © 2015 by the American Society of Nephrology.)

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway appears to be the primary regulator of muscle protein synthesis. A variety of stimuli including resistance exercise, amino acids, and hormonal signals activate mTORC1 signaling. The purpose of this study was to investigate the effect of a protein supplement on mTORC1 signaling following a resistance exercise protocol designed to promote elevations in circulating hormone concentrations. We hypothesized that the protein supplement would augment the intramuscular anabolic signaling response. Ten resistance-trained men (age, 24.7 ± 3.4 years; weight, 90.1 ± 11.3 kg; height, 176.0 ± 4.9 cm) received either a placebo or a supplement containing 20 g protein, 6 g carbohydrates, and 1 g fat after high-volume, short-rest lower-body resistance exercise. Blood samples were obtained at baseline, immediately, 30 minutes, 1 hour, 2 hours, and 5 hours after exercise. Fine-needle muscle biopsies were completed at baseline, 1 hour, and 5 hours after exercise. Myoglobin, lactate dehydrogenase, and lactate concentrations were significantly elevated after resistance exercise (P < .0001); however, no differences were observed between trials. Resistance exercise also elicited a significant insulin, growth hormone, and cortisol response (P < .01); however, no differences were observed between trials for insulin-like growth factor-1, insulin, testosterone, growth hormone, or cortisol. Intramuscular anabolic signaling analysis revealed significant elevations in RPS6 phosphorylation after resistance exercise (P = .001); however, no differences were observed between trials for signaling proteins including Akt, mTOR, p70S6k, and RPS6. The endocrine response and phosphorylation status of signaling proteins within the mTORC1 pathway did not appear to be altered by ingestion of supplement after resistance exercise in resistance-trained men., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Obesity is increasing, yet despite the necessity of maintaining muscle mass and function with age, the effect of obesity on muscle protein turnover in older adults remains unknown. Eleven obese (BMI 31.9 ± 1.1 kg · m(-2)) and 15 healthy-weight (BMI 23.4 ± 0.3 kg · m(-2)) older men (55-75 years old) participated in a study that determined muscle protein synthesis (MPS) and leg protein breakdown (LPB) under postabsorptive (hypoinsulinemic-euglycemic clamp) and postprandial (hyperinsulinemic hyperaminoacidemic-euglycemic clamp) conditions. Obesity was associated with systemic inflammation, greater leg fat mass, and patterns of mRNA expression consistent with muscle deconditioning, whereas leg lean mass, strength, and work done during maximal exercise were no different. Under postabsorptive conditions, MPS and LPB were equivalent between groups, whereas insulin and amino acid administration increased MPS in only healthy-weight subjects and was associated with lower leg glucose disposal (LGD) (63%) in obese men. Blunting of MPS in the obese men was offset by an apparent decline in LPB, which was absent in healthy-weight subjects. Lower postprandial LGD in obese subjects and blunting of MPS responses to amino acids suggest that obesity in older adults is associated with diminished muscle metabolic quality. This does not, however, appear to be associated with lower leg lean mass or strength., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Background: The slow digestion and amino acid absorption kinetics of isolated micellar casein have been held responsible for its relatively lower postprandial muscle protein synthetic response compared with rapidly digested proteins such as isolated whey. However, casein is normally consumed within a milk matrix. We hypothesized that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response after micellar casein ingestion are modulated by the milk matrix., Objective: The aim of this study was to determine the impact of a milk matrix on casein protein digestion and absorption kinetics and postprandial muscle protein synthesis in older men., Methods: In a parallel-group design, 32 healthy older men (aged 71 ± 1 y) received a primed continuous infusion of L-[ring-(2)H5]-phenylalanine, L-[ring-3,5-(2)H2]-tyrosine, and L-[1-(13)C]-leucine, and ingested 25 g intrinsically L-[1-(13)C]-phenylalanine and L-[1-(13)C]-leucine labeled casein dissolved in bovine milk serum (Cas+Serum) or water (Cas). Plasma samples and muscle biopsies were collected in the postabsorptive state and for 300 min in the postprandial period to examine whole-body and skeletal muscle protein metabolism., Results: Casein ingestion increased plasma leucine and phenylalanine concentrations and L-[1-(13)C]-phenylalanine enrichments, with a more rapid rise after Cas vs. Cas+Serum. Nonetheless, dietary protein-derived phenylalanine availability did not differ between Cas+Serum (47 ± 2%, mean ± SEM) and Cas (46 ± 3%) when assessed over the 300-min postprandial period (P = 0.80). The milk matrix did not modulate postprandial myofibrillar protein synthesis rates from 0 to 120 min (0.038 ± 0.005 vs. 0.031 ± 0.007%/h) or from 120 to 300 min (0.052 ± 0.004 vs. 0.067 ± 0.005%/h) after Cas+Serum vs. Cas. Similarly, no treatment differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed at 120 min (0.003 ± 0.001 vs. 0.002 ± 0.001) or 300 min (0.015 ± 0.002 vs. 0.016 ± 0.002 mole percent excess) after Cas+Serum vs. Cas., Conclusions: Casein ingestion in a milk matrix delays protein digestion and absorption but does not modulate postprandial muscle protein synthesis when compared to the ingestion of micellar casein only in healthy older men. This trial was registered at Nederlands Trial Register as NTR4429., (© 2015 American Society for Nutrition.)

Dysregulated anabolic responses to nutrition/exercise may contribute to sarcopenia; however, these characteristics are poorly defined in female populations. We determined the effects of two feeding regimes in older women (66 ± 2.5 yr; n = 8/group): bolus whey protein (WP-20 g) or novel low-dose leucine-enriched essential amino acids (EAA) [LEAA; 3 g (40% leucine)]. Using [(13)C6]phenylalanine infusions, we quantified muscle (MPS) and albumin (APS) protein synthesis at baseline and in response to both feeding (FED) and feeding plus exercise (FED-EX; 6 × 8 knee extensions at 75% 1-repetition maximum). We also quantified plasma insulin/AA concentrations, whole leg (LBF)/muscle microvascular blood flow (MBF), and muscle anabolic signaling by phosphoimmunoblotting. Plasma insulinemia and EAA/aemia were markedly greater after WP than LEAA (P < 0.001). Neither LEAA nor WP modified LBF in response to FED or FED-EX, whereas MBF increased to a similar extent in both groups only after FED-EX (P < 0.05). In response to FED, both WP and LEAA equally stimulated MPS 0-2 h (P < 0.05), abating thereafter (0-4 h, P > 0.05). In contrast, after FED-EX, MPS increased at 0-2 h and remained elevated at 0-4 h (P < 0.05) with both WP and LEAA. No anabolic signals quantifiably increased after FED, but p70 S6K1 Thr(389) increased after FED-EX (2 h, P < 0.05). APS increased similarly after WP and LEAA. Older women remain subtly responsive to nutrition ± exercise. Intriguingly though, bolus WP offers no trophic advantage over LEAA., (Copyright © 2015 the American Physiological Society.)

Alcohol abuse, either by acute intoxication or prolonged excessive consumption, leads to pathological changes in many organs and tissues including skeletal muscle. As muscle protein serves not only a contractile function but also as a metabolic reserve for amino acids, which are used to support the energy needs of other tissues, its content is tightly regulated and dynamic. This review focuses on the etiology by which alcohol perturbs skeletal muscle protein balance and thereby over time produces muscle wasting and weakness. The preponderance of data suggest that alcohol primarily impairs global protein synthesis, under basal conditions as well as in response to several anabolic stimuli including growth factors, nutrients, and muscle contraction. This inhibitory effect of alcohol is mediated, at least in part, by a reduction in mTOR kinase activity via a mechanism that remains poorly defined but likely involves altered protein-protein interactions within mTOR complex 1. Furthermore, alcohol can exacerbate the decrement in mTOR and/or muscle protein synthesis present in other catabolic states. In contrast, alcohol-induced changes in muscle protein degradation, either global or via specific modulation of the ubiquitin-proteasome or autophagy pathways, are relatively inconsistent and may be model dependent. Herein, changes produced by acute intoxication versus chronic ingestion are contrasted in relation to skeletal muscle metabolism, and limitations as well as opportunities for future research are discussed. As the proportion of more economically developed countries ages and chronic illness becomes more prevalent, a better understanding of the etiology of biomedical consequences of alcohol use disorders is warranted., (Copyright © 2015 the American Physiological Society.)

High-S (HS) diets have been identified as a causative agent in the development of oxidative stress in cattle, which in postmortem muscle can negatively alter meat quality. Vitamin C (VC) is a potent antioxidant produced endogenously by cattle; however, exogenous supplementation of VC may be useful when HS diets are fed to cattle. The objective of this study was to examine the impact of duration of VC supplementation, for the first 56, 90, or 127 d, during the finishing period on meat color and tenderness of the longissimus thoracis (LT) collected from calf-fed steers consuming a 0.31 or 0.59% S diet. Angus steers ( n= 42) were stratified to pens by initial BW (304 ± 13 kg) and GeneMax marbling score (4.3 ± 0.12), and each pen was randomly assigned to 1 of 7 treatments (6 steers/pen, 1 pen/treatment), including HS (0.59% S, a combination of dried distillers grains plus solubles and sodium sulfate) control (HS CON), HS CON + 10 g VC·steer·(-1)d(-1) for the first 56 d (HS VC56), 90 d (HS VC90), or 127 d (HS VC127), low S (LS; 0.31% S) + 10 g VC·steer·(-1)d(-1) for the first 56 d (LS VC56), 90 d (LS VC90), or 127 d (LS VC127). Steers were harvested (n = 40) and, after a 24-h chill, rib sections (LT) were collected. pH was determined on each rib section before division into 3 sections for determination of 1) 7-d retail display and color and Warner-Bratzler shear force (WBSF), 2) 14-d WBSF determination, and 3) protein degradation and collagen content (2 d postmortem). Data were analyzed by ANOVA as a completely randomized design, with the fixed effect of treatment. Individual feed intake was recorded, and steer was the experimental unit. The HS steers had a greater and lesser percent of the 80- and 76-kDa subunits of calpain-1 (P ≤ 0.05), respectively, and tended to have less (P = 0.08) troponin T degradation (d2), and more (P = 0.02) collagen than LS steers. Increasing days of VC supplementation decreased (P = 0.05) the percentage of the 80 kDa subunit of calpain-1 in HS steers but actually increased it in LS steers (P= 0.003). Supplementing VC, regardless of dietary S, did not affect meat collagen, WBSF, or color (P ≥ 0.12). a* and b* values were greater (P ≤ 0.05) in the LS treatments compared to the HS treatments. Increasing the days of VC supplementation to steers fed a HS diet appears to alleviate the negative effects of the HS diet on calpain-1 but has no effect on muscle tenderness or meat color.

Strategies to enhance weight loss with a high fat-to-lean ratio in overweight/obese older adults are important since lean loss could exacerbate sarcopenia. We examined how dietary protein distribution affected muscle protein synthesis during energy balance (EB), energy restriction (ER), and energy restriction plus resistance training (ER + RT). A 4-wk ER diet was provided to overweight/obese older men (66 ± 4 yr, 31 ± 5 kg/m(2)) who were randomized to either a balanced (BAL: 25% daily protein/meal × 4) or skewed (SKEW: 7:17:72:4% daily protein/meal; n = 10/group) pattern. Myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR) were measured during a 13-h primed continuous infusion of l-[ring-(13)C6]phenylalanine with BAL and SKEW pattern of protein intake in EB, after 2 wk ER, and after 2 wk ER + RT. Fed-state myofibrillar FSR was lower in ER than EB in both groups (P < 0.001), but was greater in BAL than SKEW (P = 0.014). In ER + RT, fed-state myofibrillar FSR increased above ER in both groups and in BAL was not different from EB (P = 0.903). In SKEW myofibrillar FSR remained lower than EB (P = 0.002) and lower than BAL (P = 0.006). Fed-state sarcoplasmic protein FSR was reduced similarly in ER and ER + RT compared with EB (P < 0.01) in both groups. During ER in overweight/obese older men a BAL consumption of protein stimulated the synthesis of muscle contractile proteins more effectively than traditional, SKEW distribution. Combining RT with a BAL protein distribution "rescued" the lower rates of myofibrillar protein synthesis during moderate ER., (Copyright © 2015 the American Physiological Society.)

Objective: The aim of this study was to investigate the effects of alanyl-glutamine (Ala-Gln) on skeletal muscle protein synthetic and degradative signaling in piglets challenged with Escherichia coli lipopolysaccharide (LPS)., Methods: Piglets were arranged in a 2 × 2 factorial design and the main effects were LPS challenge (0 or 100 units) and diets (0.62% Ala or 0.5% Ala-Gln). After treatment with either Ala or Ala-Gln for 10 d, piglets were injected twice with either saline or LPS on days 11 and 15., Results: During days 11 to 15 (postchallenge), LPS challenge affected the growth performance of piglets. Ala-Gln supplementation tended to alleviate the reduction of the average daily weight gain (P = 0.071) and the average daily feed intake (P = 0.087) of the LPS-challenged piglets. LPS challenge increased the concentrations of cytokines in plasma (P < 0.05), however, Ala-Gln supplementation prevented the elevation of cortisol induced by LPS challenge (P < 0.05). Moreover, Ala-Gln supplementation increased the mRNA expressions of insulin-like growth factor-1 signaling and Akt (P < 0.05). Ala-Gln supplementation also increased the phosphorylation abundance of the mammalian target of rapamycin, eIF-4 E binding protein 1 and ribosomal protein S6 kinase 1 (P < 0.05). Additionally, Ala-Gln supplementation down-regulated the mRNA abundances of toll-like receptor 4 (TLR4), muscle atrophy F-box, and muscle RING finger 1, which are associated with protein degradation induced by LPS challenge., Conclusion: Ala-Gln supplementation had beneficial effects in improving protein synthesis signaling of skeletal muscle, and reversed the deleterious changes of signaling molecules in muscle atrophy mainly through down-regulation of Akt/FOXO and TLR4 signaling pathways induced by LPS challenge., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Context: Androgen-deprivation therapy (ADT) is a key component of treatment for aggressive and advanced prostate cancer, but it has also been associated with adverse effects on bone, metabolic, cardiovascular, sexual, and cognitive health as well as body composition., Objective: To review the current literature on the adverse effects of ADT and strategies for ameliorating harm from ADT., Evidence Acquisition: The Medline database (through PubMed) was searched from inception to August 1, 2013, for studies documenting the side effects of ADT and for randomized and prospective trials of interventions to mitigate those side effects., Evidence Synthesis: Adverse effects of ADT include decreases in bone mineral density; metabolic changes such as weight gain, decreased muscle mass, and increased insulin resistance; decreased libido and sexual dysfunction; hot flashes; gynecomastia; reduced testicle size; anemia; and fatigue. Several observational studies suggest an increased risk of diabetes and cardiovascular events, although most published studies report that ADT is not linked to greater cardiovascular mortality. Randomized trials have found value in treatments for some adverse effects including bone loss (bisphosphonates, denosumab, selective estrogen receptor modulators), markers of metabolic syndrome (exercise, diet, metformin), gynecomastia (tamoxifen, prophylactic radiation), muscle loss (resistance and aerobic exercise), and hot flashes (venlafaxine, medroxyprogesterone, cyproterone acetate, gabapentin)., Conclusions: ADT is often a necessary component of the treatment of aggressive prostate cancer, yet it has known harms that can impair health and quality of life. Clinicians should be aware of interventions that can help mitigate these adverse effects., Patient Summary: Androgen deprivation therapy is a critical component of the management of aggressive and advanced prostate cancer, but it causes adverse effects including bone loss, metabolic changes, gynecomastia, muscle loss, hot flashes, and possibly increased cardiovascular events. Clinicians should be aware of interventions that can help mitigate these adverse effects., (Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

In the present study, we performed a meta-analysis to assess the ability of leucine supplementation to increase the muscle protein fraction synthetic rate and to augment lean body mass or leg lean mass in elderly patients. A literature search was conducted on Medline, Cochrane, EMBASE and Google Scholar databases up to 31 December 2013 for clinical trials that investigated the administration of leucine as a nutrient that affects muscle protein metabolism and muscle mass in elderly subjects. The included studies were randomised controlled trials. The primary outcome for the meta-analysis was the protein fractional synthetic rate. Secondary outcomes included lean body mass and leg lean mass. A total of nine studies were included in the meta-analysis. The results showed that the muscle protein fractional synthetic rate after intervention significantly increased in the leucine group compared with the control group (pooled standardised difference in mean changes 1·08, 95% CI 0·50, 1·67; P< 0·001). No difference was found between the groups in relation to lean body mass (pooled standardised difference in mean changes 0·18, 95% CI - 0·18, 0·54; P= 0·318) or leg lean mass (pooled standardised difference in mean changes 0·006, 95% CI - 0·32, 0·44; P= 0·756). These findings suggest that leucine supplementation is useful to address the age-related decline in muscle mass in elderly individuals, as it increases the muscle protein fractional synthetic rate.

We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR)-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (AR(ΔZF2)) versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR(∆ZF2) muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57(Kip2), Igf2 and calcineurin Aa, was increased in AR(∆ZF2) muscle, and the expression of all but p57(Kip2) was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

Recent studies in mice and zebrafish suggest that the unannotated Src homology 3 and cysteine rich domain 3 (STAC3) gene plays an important role in skeletal muscle development and contraction. The objective of this study was to determine the tissue specificity of the bovine STAC3 gene and its potential role in the proliferation and differentiation of bovine satellite cells. The STAC3 mRNA was detected only in skeletal muscle among 18 bovine tissues examined by reverse transcription PCR. Western blotting revealed the expression of STAC3 protein in bovine skeletal muscle and the absence of it in 6 bovine tissues analyzed. Transfection of the bovine satellite cells with a pool of 2 STAC3 small interfering RNA (siRNA) caused a 90% reduction in STAC3 mRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. Approximately 60% of bovine satellite cells transfected with STAC3 siRNA formed myotubes by 72 h of differentiation, whereas that percentage was 40% for those transfected with negative control siRNA (P < 0.05). At 24, 48, and 72 h of differentiation, bovine satellite cells transfected with STAC3 siRNA had greater mRNA expression of myogenin, myosin heavy chain 3, and myosin heavy chain 7, markers of myotubes, than those transfected with negative control siRNA (P < 0.05). These results suggest that the STAC3 gene is a negative regulator of the differentiation and fusion of bovine satellite cells into myotubes. However, STAC3 expression was increased during the differentiation of bovine satellite cells into myotubes. This suggests that STAC3 might have different functions in bovine myotubes than in bovine satellite cells.