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Regulation of atrogin-1 and protein degradation following incubation with dexamethasone and
α in mouse C2C12 and primary human myotubes
A.E. Larsen, T.C. Crowe and A.P. Russell, The Centre for Physical Activity and Nutrition Research (C-PAN),
School of Exercise and Nutrition Sciences, Deakin University, VIC 3125, Australia.

Introduction: Atrogin-1 is a muscle specific E3-ligase involved in muscle wasting (Bodine et al., 2001).
Increased levels of atrogin-1 mRNA has been observed in numerous in vitro and in vivo rodents models ofmuscle atrophy (Glass, 2005). Human studies performed have shown that atrogin-1 is increased in humanatrophy conditions, such as leg immobilization (Jones et al., 2004), ALS (Leger et al., 2006), COPD (Doucet etal., 2007) and quadriplegic myopathy (Di Giovanni et al., 2004). In mice, but not humans, fasting increasesatrogin-1 (Sandri et al., 2004; Larsen et al., 2006), suggesting that species differences may exists with respect toits regulation. The aim of the present study was to determine the regulation of atrogin-1 and protein degradationfollowing treatment with dexamethasone (DEXA) and TNFα in mouse C2C12 and human primary myotubes.
Methods: Mouse C2C12 myotubes and primary human myotubes were treated with either TNF-α (20
ng/mL) or DEXA (10 µM) for 1, 4, 24 and 48-h. Atrogin-1 mRNA lev els were measured using real time-PCR.
Protein degradation was determined by measuring the release of [3H]-tyrosine into the media.
Results: Atrogin-1 mRNA was significantly increased 2- and 4-fold in C2C12 myotubes after 24 and 48-h
treatment with DEXA, respectively. In human myoblasts atrogin-1 was increased 2.2-fold only after 48-h ofDEXA treatment. After treating C2C12 cells with TNF-α, atrogin-1 showed a transient change, increasing by50% following 1-hr of treatment, decreasing to 50% below control levels following 4h of treatment thenretuning to control levels after 24 and 48-h. In contrast, human myotubes treated with TNF-α showed a 3.1 foldincrease in atrogin-1 after 48-h of treatment. In the human myotubes the increase in atrogin-1 mRNA lev elsfollowing 48-h of both DEXA and TNF-α treatment resulted in significant increases in protein degradation byapproximately 15%.
Conclusions: Treatment of both mouse C12C12 myotubes and primary human myotubes with DEXA
results in increases in atrogin-1 mRNA; however human myotubes require a longer treatment period. Treatmentwith TNF-α demonstrated a more dramatic species dependent effect, with mouse C2C12 myotubes presenting arapid increase, then decrease in atrogin-1 over 1-4 h of treatment, followed by a return to baseline lines at 48-h.
In contrast, human myotubes had an increase in atrogin-1 mRNA 48- after treatment. In human myotubes theincreases in atrogin-1 caused by both DEXA and TNF-α was associated with an increase in protein degradation.
These observations highlight the need for caution when translating results obtained in rodent models to humanconditions.
Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM Stitt, TN Yancopoulos GD, Glass DJ. (2001).
Science, 294: 1704-8.
Di Giovanni S, Molon A, Broccolini A, Melcon G, Mirabella M, Hoffman EP & Servidei S. (2004). Annals of Neurology, 55: 195-206.
Doucet M, Russell AP, Leger B, Debigare R, Joanisse DR, Caron MA, Leblanc P & Maltais F. (2007). American Journal of Respiratory and Critical Care Medicine, 176: 261-9.
Glass DJ. (2005). International Journal of Biochemistry and Cell Biology, 37: 1974-84.
Jones SW, Hill RJ, Krasney PA, O’Conner B, Peirce N, Greenhaff PL. (2004) FASEB Journal, 18: 1025-7.
Larsen AE, Tunstall RJ, Carey KA, Nicholas G, Kambadur R, Crowe TC, Cameron-Smith D. (2006). Annals of
Nutrition and Metabolism, 50: 476-81.
Leger B, Vergani L, Soraru G, Hespel P, Derave W, Gobelet C, D’Ascenzio C, Angelini C, Russell AP. (2006).
FASEB Journal, 20: 583-5.
Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL. (2004) Cell, 117: 399-412.
Proceedings of the Australian Physiological Society


Microsoft word - manual therapy magical trick or science.doc

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