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Title: The role of resveratrol and Sirtuin1 in skeletal muscle under a nutrient stress
Author: Dugdale, H. F.
Awarding Body: Liverpool John Moores University
Current Institution: Liverpool John Moores University
Date of Award: 2017
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Abstract:
Dietary restriction (DR) is the only known nutritional manipulation that can increase both lifespan and healthspan in a variety of species. Underlying these increases are improvements in metabolic health and reductions in cancer incidence. Despite these physiological improvements, the regulation of skeletal muscle mass is extremely sensitive to alterations in nutrients [reviewed in (Sharples et al., 2015)] and as such has been reported to reduce regenerative potential and increase atrophy in skeletal muscle cells and myotubes. Interestingly, the activation of Sirtuin1 (SIRT1) has been reported during DR and its reduction abrogates lifespan extension. Importantly, SIRT1 activation via resveratrol treatment has been indicated to be important in the presence of inflammatory stress (TNF-α) (Saini et al., 2012). Resveratrol supplementation has also improved survival and regeneration of skeletal muscle cells as well in muscle cell remodelling following oxidative stress (Bosutti and Degens, 2015). We therefore sought to create an in-vitro physiological model of DR by mimicking levels of glucose in the circulation and interstitium in-vivo in response to DR (Chapter 3) as well as optimising the activation and inhibition of SIRT1 using resveratrol and SIRT1 inhibitor, EX-527 respectively (Chapter 4). With our ultimate aim to investigate the potential role and mechanisms of the activation/inhibition of SIRT1 in ameliorating the degenerative/atrophic effect of DR in both differentiating myoblasts (Chapter 5) and mature myotubes (Chapter 6). Indeed, in Chapter 3 we present two models of reduced glucose; one reduced (medium/ MED) and the other blocked (LOW) differentiation and myotube hypertrophy. The former represented circulatory glucose blood levels (MED 1.13 g/L or 6.25 mM) and the latter interstitial represented glucose levels (LOW 0.56 g/L or 3.12 mM) of rodents under DR. In Chapter 4 we also suggest that within the in vitro muscle cell model, activation/inhibition of SIRT1 phosphorylation (western blot analysis) was thought to be most effective at 10 μM of resveratrol and 100 nM of EX-527 respectively. In chapter 5, we observed that resveratrol treatment did not improve fusion when administered to differentiating myoblasts. Resveratrol did however evoke increases in myotube hypertrophy under normal glucose conditions. Importantly resveratrol enabled improved myotube hypertrophy over an acute 24 h period when administered to existing mature myotubes in low glucose environments. If this finding translates to whole organisms and human populations it could provide healthspan improvements via reductions in fragility associated with loss of muscle mass in individuals undergoing dietary restriction. After this 24 h period resveratrol was unable to reduce myotube atrophy and the myotubes continued to atrophy, suggestive of a need for repeated resveratrol treatment to enable continued protection against muscle atrophy under low glucose conditions. SIRT1 activation increased Myogenic regulatory factor 4 (MRF4) gene expression under LOW glucose conditions which was associated with the observed improvements in myotube size at 24 h. Whereas, SIRT1 activation via resveratrol treatment in normal glucose conditions modulated increased gene expression of Myosin heavy chain 7 (MYHC7) coding for the slow isoform while inhibition of SIRT1 (EX-527) lead to reductions in gene expression of MYHC 1, 2 and 4, coding for faster IIx, IIa, IIb isoforms respectively. Perhaps suggesting that elevated SIRT1 was important in the activation of genes coding for slower myosin heavy chain isoforms. Furthermore, while SIRT activation via resveratrol did modulate increases in IGF-I gene expression, it did not appear to modulate energy sensing AMP activated protein kinase (AMPK) vs. growth related Protein 70 S6 Kinase (p70S6K) signalling pathways. However, SIRT1 inhibition increased AMPK activity in both low and normal glucose with corresponding mean reductions in p70S6K in normal glucose conditions. This indicates that perhaps normal SIRT1 activity was required for appropriate AMPK activation, which may therefore prevent the suppression of p70S6K and the corresponding reductions in myotube size observed in SIRT1 inhibitor conditions. Furthermore, during low glucose induced myotube atrophy resveratrol reduced gene expression of the negative regulator of muscle mass, myostatin and protein degradative ubiquitin ligase enzyme, MUSA1. Overall, SIRT1 activation via a single dose of resveratrol appears to have a role in acutely negating the effect of low glucose induced myotube atrophy and promoting myotube hypertrophy when glucose is readily available.
Supervisor: sharples, A. P. ; Stewart, C. E. ; morton, J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.718880  DOI: Not available
Keywords: TX341 Nutrition. Foods and food supply ; QP Physiology
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