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NMR Based Metabonomics Study of DAG Treatment in a C2C12 Mouse Skeletal Muscle Cell Line Myotube Model of Burn-Injury

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Abstract

After severe burn injury, proinflammatory cytokine levels are elevated in serum and skeletal muscle, which in turn increases protein breakdown and decreases protein synthesis. In this study, C2C12 mouse skeletal muscle cell line myotubes were exposed to proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) as an in vitro cell-line model of catabolic response to burn injury and then treated with des-acyl ghrelin (DAG), a 28 amino acid polypeptide hormone thought to inhibit protein breakdown and increase protein synthesis, to assess its therapeutic potential. Nuclear magnetic resonance-based metabonomics was used to monitor metabolic activity of C2C12 myotubes under four treatment conditions: (1) control, (2) TNF-α/IFN-γ (TI), (3) DAG (DA), and (4) TNF-α/IFN-γ followed by DAG (TIDA) to assess the effect of DAG treatment on cellular metabolic response during basal or catabolic conditions. Twelve metabolites showed significant changes in concentrations following treatments in the hydrophilic cell extracts. Lactate (P < 10−4) and citrulline (P < 10−9) increased with TNF-α/IFN-γ treatment, indicating increased protein degradation, and returned to control levels in the TIDA group. Adenosine nucleotide levels had decreased trends in TI myotubes that returned to baseline levels after DAG treatment (P < 10−4). Guanidinoacetate and pantothenate, metabolites involved in protein synthesis and cell proliferation, had increased concentration trends following DAG treatment in both the DA and TIDA groups. Our metabonomics analysis provides further evidence that DAG counteracts the catabolic response caused by elevated muscle TNF-α/IFN-γ cytokine levels following severe burns and can play a potential therapeutic role in treatment of burn injury.

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Abbreviations

NMR:

Nuclear magnetic resonance

PCA:

Principal component analysis

DAG:

Des-acyl ghrelin

TNF-α:

Tumor necrosis factor-α

IFN-γ:

Interferon-γ

TI:

TNF-α/IFN-γ treated group

DA:

DAG treated group

TIDA:

TNF-α/IFN-γ followed by DAG treated group

TI/TIDA:

Comparison between TI and TIDA group

EDL:

Extensor digitorum longus

IGF-I:

Insulin-like growth factor-I

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Acknowledgments

The authors would like to acknowledge support of Miami University and the Ohio Board of Regents for funding to establish the Ohio Eminent Scholar Laboratory where the work was performed. This study is supported in part by Shriners Hospital for Children grant # 864006 (AB) and a Department of Veteran Affairs Grant #1I01BX000263 (AB). The authors would also like to acknowledge support from Bruker Biospin, Inc that enabled development of the statistical significance analysis software used in the analysis of the data reported in this paper. MW would like to acknowledge support of a Miami University Cell, Molecular, and Structural Biology Fellowship.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Miami University, 106 Hughes Laboratories, Oxford, OH, 45056, USA

    Miki Watanabe, Theresa A. Ramelot, Junho Cho, Kenneth B. Lewis & Michael A. Kennedy

  2. Department of Surgery, University of Cincinnati Medical Center, Cincinnati, OH, 45267, USA

    Sulaiman Sheriff & Ambikaipakan Balasubramaniam

  3. Shriners Hospital for Children, Cincinnati, OH, 45229, USA

    Nijiati Kadeer & Ambikaipakan Balasubramaniam

  4. Cincinnati Veterans Affairs Medical Center, Cincinnati, OH, 45220, USA

    Ambikaipakan Balasubramaniam

Authors
  1. Miki Watanabe
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  2. Sulaiman Sheriff
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  3. Theresa A. Ramelot
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  4. Nijiati Kadeer
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  5. Junho Cho
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  6. Kenneth B. Lewis
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  7. Ambikaipakan Balasubramaniam
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  8. Michael A. Kennedy
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Correspondence to Michael A. Kennedy.

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Miki Watanabe and Sulaiman Sheriff contributed equally to this work.

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Watanabe, M., Sheriff, S., Ramelot, T.A. et al. NMR Based Metabonomics Study of DAG Treatment in a C2C12 Mouse Skeletal Muscle Cell Line Myotube Model of Burn-Injury. Int J Pept Res Ther 17, 281 (2011). https://doi.org/10.1007/s10989-011-9264-x

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