L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

Christopher Brooks Mobley; Carlton D Fox; Brian S Ferguson; Rajesh H Amin; Vincent J Dalbo; Shawn Baier; John A Rathmacher; Jacob M Wilson; Michael D Roberts

doi:10.1186/1550-2783-11-38

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

J Int Soc Sports Nutr. 2014 Aug 13:11:38. doi: 10.1186/1550-2783-11-38. eCollection 2014.

Authors

Christopher Brooks Mobley¹, Carlton D Fox¹, Brian S Ferguson¹, Rajesh H Amin², Vincent J Dalbo³, Shawn Baier⁴, John A Rathmacher⁴, Jacob M Wilson⁵, Michael D Roberts¹

Affiliations

¹ School of Kinesiology, Molecular and Applied Sciences Laboratory, Auburn University, 301 Wire Road, Office 286, Auburn, AL 36849, USA.
² Harrison School of Pharmacy, Auburn University, Auburn, AL, USA.
³ School of Medical and Applied Sciences, Central Queensland University, Rockhampton, QLD, Australia.
⁴ Metabolic Technologies, Inc, Ames, IA, USA.
⁵ Department of Health Sciences and Human Performance, University of Tampa, Tampa, FL, USA.

Abstract

Background: The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes.

Methods: After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1).

Results: MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively.

Conclusions: Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo.

Keywords: Akirin-1; Atrophy; GDF8; Myostatin; Skeletal muscle.