Leucine Elicits Myotube Hypertrophy and Enhances Maximal Contractile Force in Tissue

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  This article is protected by copyright. All rights reserved ORIGINAL RESEARCH ARTICLE   Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro †  Martin N.R.W 1,2 , Turner M.C 1,2, , Farrington, R 1 , Player D.J 1,2,3 , Lewis M.P* 1,2,3 . 1 School of Sport, Exercise and Health Sciences, Loughborough University, UK 2  National Centre for Sport and Exercise Medicine, School of Sport, Exercise and Health Sciences, Loughborough University, UK. 3 Arthritis Research UK Centre for Sport, Exercise and Osteoarthritis, School of Sport, Exercise and Health Sciences, Loughborough University, UK. *Corresponding author:Professor Mark P. Lewis  National Centre for Sport and Exercise Medicine, School of Sport, Exercise and Health Sciences, Loughborough University, UK M.P.Lewis@lboro.ac.uk Tel: 01509 226430, Fax: 01509 226347Running head: Leucine improves engineered muscle function † This article has been accepted for publication and undergone full peer review but has not  been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcp.25960]   Additional Supporting Information may be found in the online version of this article. Received 27 September 2016; Revised 12 April 2017; Accepted 13 April 2017 Journal of Cellular Physiology This article is protected by copyright. All rights reserved DOI 10.1002/jcp.25960    This article is protected by copyright. All rights reserved  Abstract The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilised a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signalling, myotube growth and muscle function.Phosphorylation of the mTORC1 target proteins 4EBP-1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20mM of leucine inducing similar effects, which were greater than those seen with 1mM. Maximal contractile force was also elevated with leucine supplementation; however although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co-incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hours) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly-controlled investigations into nutritional regulation of muscle physiology. This article is protected by copyright. All rights reserved Keyword: amino acids, mTORC1, hypertrophy, skeletal muscle    This article is protected by copyright. All rights reserved Introduction Skeletal muscle growth is regulated primarily by the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway, which enhances the capacity for mRNA translation and reduces flux through catabolic pathways such as the autophagy-lysosome and the ubiquitin proteasome system (Nicklin et al. 2009). mTORC1 signalling has consistently shown to be activated in response to both muscle loading (e.g. resistance exercise), and amino acid consumption/treatment (Marcotte et al. 2015), and as such these stimuli represent excellent candidates as therapies for attenuating the muscle wasting associated with a number of disease states and ageing. Indeed, acute human studies have observed activation of mTORC1 and its downstream targets (e.g. p70S6K, rpS6 and 4EBP-1) following ingestion of mixed amino acids, and this is coupled with an increase in muscle protein synthesis (MPS) in the ensuing 60-120 minutes (Atherton et al. 2010; Koopman et al. 2006; Paddon-Jones et al. 2004; Volpi et al. 2003). Furthermore, when human skeletal muscle undergoes loading prior to amino acid ingestion this effect on mTORC1 signalling and MPS is potentiated (Moore et al. 2011; Witard et al. 2014). The necessity for mTOR activation in mediating the MPS response to both amino acids and muscle loading is evidenced by the fact that in rodents, both stimuli fail to augment the synthetic response when in the presence of the mTOR inhibitor rapamycin (Anthony et al. 2000; Kubica et al. 2005). The anabolic properties of amino acid ingestion have been largely attributed to the essential amino acids, and in particular the branched chain amino acid leucine. A number of lines of research support this notion; firstly, whey protein, which has a  This article is protected by copyright. All rights reserved high leucine content results in superior MPS rates in humans compared to soy or casein, which have lower leucine contents (Tang et al. 2009). Secondly, ingestion of small quantities of leucine rich essential amino acids activate the downstream mTORC1 target p70S6k and MPS in a comparable manner to 20-25g of whey protein, and to a greater extent than a bolus of leucine-deficient essential amino acids (Bukhari et al. 2015; Churchward-Venne et al. 2012), and removal of leucine from an essential amino acid supplement following muscle loading attenuates mTORC1 signalling (Moberg et al. 2014). Finally, in C2C12 myotubes in vitro , leucine exhibits the most potent stimulation of mTORC1 signalling compared to all other amino acids (Atherton, Smith, et al. 2010), and its deprivation impairs protein synthesis and phosphorylation of p70S6k (Talvas et al. 2006). In vitro  cultures of skeletal muscle provide a controlled and isolated environment in which to understand cellular and molecular adaptation, and have improved our understanding of the importance of amino acids, and in particular leucine, for skeletal muscle growth (Areta et al. 2014; Atherton et al. 2010; Talvas et al. 2006). However, a limitation of conventional in vitro  methods is the inability of the rigid 2-dimensional substrate to support muscle contraction, and as such only acute experiments are typically possible. Tissue engineered skeletal muscle however allows for skeletal muscle progenitor cells to be cultured on/inside biologically relevant substrates in 3-dimensions, and are less stiff, in turn supporting improvements in levels of skeletal muscle maturation (Engler et al. 2004), and generation of contractile force. Indeed, the ability to stimulate and measure contractile force within tissue engineered skeletal muscle is well reported (Cheng et al. 2014), and whilst we and others (Martin et al. 2015; Ostrovidov et al. 2017) have made efforts towards increasing the
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