title: L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy.
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.
authors.affiliation: School of Kinesiology, Molecular and Applied Sciences Laboratory, Auburn University, 301 Wire Road, Office 286, Auburn, AL 36849, USA.
authors.forename: Christopher Brooks
authors.initials: CB
authors.lastname: Mobley
authors.affiliation: School of Kinesiology, Molecular and Applied Sciences Laboratory, Auburn University, 301 Wire Road, Office 286, Auburn, AL 36849, USA.
authors.forename: Carlton D
authors.initials: CD
authors.lastname: Fox
authors.affiliation: School of Kinesiology, Molecular and Applied Sciences Laboratory, Auburn University, 301 Wire Road, Office 286, Auburn, AL 36849, USA.
authors.forename: Brian S
authors.initials: BS
authors.lastname: Ferguson
authors.affiliation: Harrison School of Pharmacy, Auburn University, Auburn, AL, USA.
authors.forename: Rajesh H
authors.initials: RH
authors.lastname: Amin
authors.affiliation: School of Medical and Applied Sciences, Central Queensland University, Rockhampton, QLD, Australia.
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Dalbo
authors.affiliation: Metabolic Technologies, Inc, Ames, IA, USA.
authors.forename: Shawn
authors.initials: S
authors.lastname: Baier
authors.affiliation: Metabolic Technologies, Inc, Ames, IA, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Rathmacher
authors.affiliation: Department of Health Sciences and Human Performance, University of Tampa, Tampa, FL, USA.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Wilson
authors.affiliation: School of Kinesiology, Molecular and Applied Sciences Laboratory, Auburn University, 301 Wire Road, Office 286, Auburn, AL 36849, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Roberts
keywords: Akirin-1; Atrophy; GDF8; Myostatin; Skeletal muscle