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Fenugreek increases insulin-stimulated creatine content in L6C11 muscle myotubes

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Abstract

Purpose

Creatine uptake by muscle cells is increased in the presence of insulin. Accordingly, compounds with insulin-like actions may also augment creatine uptake. The aim of this study was to investigate whether Trigonella foenum-graecum (fenugreek), an insulin mimetic, increases total intracellular creatine levels in vitro.

Methods

Total cellular creatine content was measured fluorometrically in L6C11 muscle myotubes treated for 1, 4, and 24 h with 0.5 mM creatine (CR), CR and 20 μg/mL fenugreek seed extract (CR + FEN), CR and 100 nM insulin (CR + INS), and CR + INS + FEN (n = 6 per treatment group). Alterations in the expression of the sodium- and chloride-dependent creatine transporter, SLC6A8, and key signaling proteins in the PI3-K/Akt pathway were determined.

Results

Compared to control (CON), CR + INS + FEN increased total creatine content after 4 h (P < 0.05), whereas all conditions increased SLC6A8 protein expression above CON at this time (P < 0.05). Changes in insulin signaling were demonstrated via increases in AktThr308 phosphorylation, with CR + INS > CON and CR at 1 h (P < 0.05) and with CR + INS + FEN > CON, CR, and CR + INS at 4 h (P < 0.05). In contrast, no changes in PKCζ/λ or GLUT4 phosphorylation were detected.

Conclusion

Fenugreek, when combined with insulin, modulates creatine content via a mechanism which is independent of the activity of SLC6A8, suggesting that an alternative mechanism is responsible for the regulation and facilitation of insulin-mediated creatine uptake in skeletal muscle cells.

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Acknowledgments

The authors of this paper would like to thank A/Prof. Theo Macrides and the RMIT Natural Products Research Group (Dr. Lynn Hodges, Mr. Donald Harney, and Ms. Nicolette Kalafatis) as well as Mr. Robert Brkljaca for their invaluable help in the extraction process. We appreciate and thank Mr. Frank Antolasic for the analysis of the fenugreek extract samples. Additionally, we would like to thank Mr. Griffin D’costa for his input and valuable discussions about fenugreek and cell culture technique.

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Authors and Affiliations

  1. Centre for Exercise and Nutrition, Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, VIC, 3065, Australia

    Kristyen A. Tomcik, William J. Smiles, Donny M. Camera, John A. Hawley & Rani Watts

  2. School of Applied Sciences and Health Innovations Research Institute, RMIT University, Melbourne, VIC, Australia

    Helmut M. Hügel

  3. Research Institute for Sports and Exercise Sciences, Liverpool John Moores University, Liverpool, UK

    John A. Hawley

Authors
  1. Kristyen A. Tomcik
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  2. William J. Smiles
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Correspondence to John A. Hawley.

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Figure 1

A liquid chromatography-mass spectroscopy (LC-MS) method was developed and validated for the analysis of (2S,3R,4S)- 4-hydroxyisoleucine (4-HIL) in fenugreek seed extract. A- A representative analytical scale of separation and abundance for fenugreek seed extract run on an Electrospray Ionization ESI mass spectrometer. Extract analysis was performed by monitoring the area under the 4-HIL peak at mass/charge of 148 which corresponds to the [4HIL+1] parent ion in the mass spectrum in each extract sample. B- A comparison of the percentage of 4-HIL per extract by weight. A calibration curve was created from 4-HIL standards and fenugreek extract 4-HIL samples. The weight of the 4-HIL content from each fenugreek extract sample was determined using area under the curve and calculated as a percentage. (JPEG 62 kb)

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Tomcik, K.A., Smiles, W.J., Camera, D.M. et al. Fenugreek increases insulin-stimulated creatine content in L6C11 muscle myotubes. Eur J Nutr 56, 973–979 (2017). https://doi.org/10.1007/s00394-015-1145-1

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  • DOI: https://doi.org/10.1007/s00394-015-1145-1

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