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. 2018 Apr 3;8(1):5525.
doi: 10.1038/s41598-018-23891-5.

Proteolysis Inhibition by Hibernating Bear Serum Leads to Increased Protein Content in Human Muscle Cells

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Free PMC article

Proteolysis Inhibition by Hibernating Bear Serum Leads to Increased Protein Content in Human Muscle Cells

Stéphanie Chanon et al. Sci Rep. .
Free PMC article

Abstract

Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Winter bear serum promotes an increase in human muscle cell size. Illustrative immunodetection and corresponding quantification of myosin heavy chain in cultured myotubes upon standard culture condition with fetal bovine serum (FBS), winter bear serum (WBS) or summer bear serum (SBS) treatment. Results are the mean ± SEM of 6 independent experiments (different cell preparations and bear serum mixes). Scale bar: 100 µm. Significant differences result from paired T-tests (*p < 0.05).
Figure 2
Figure 2
Winter bear serum inhibits protein degradation and synthesis rates in human muscle cells. Overall protein degradation rates in human myotubes exposed for 24 hours to FBS, SBS or WBS (A), and rates of proteolysis after proteasomal or lysosomal inhibition (B) were measured. For the latter, twenty-four hours after serum exposure, inhibitors (1 µM Bortezomid [PS-341], 0.1 µM concanamycin [Conc. A], or DMSO only [Veh.]) were added, and rates of proteolysis were determined for six hours. Proteasomal and lysosomal degradation rates were expressed as the difference between total and specifically inhibited rates. Overall protein synthesis rates were assessed in human myotubes with quantification of puromycin incorporation after 48 hours of exposure to FBS, SBS or WBS (C,D). Protein synthesis rates were also assessed by measuring [3H]-tyrosine incorporation in human myotubes exposed for 48 hours to SBS or WBS (E). (F) Shows incorporation rates of [3H]-tyrosine and puromycin after 24 hours. Results are the mean ± SEM of at least 3 independent experiments measured in duplicate. Significant differences result from paired T-tests (*p < 0.05; **p < 0.01).
Figure 3
Figure 3
Winter bear serum inhibits proteasomal degradation in human muscle cells. Illustrative immunodetection (A) and corresponding quantification (B) of ubiquitinated proteins in human myotubes exposed for 24 hours to FBS, SBS or WBS. Quantifications were performed after one hour of proteasomal (1 µM Bortezomid [PS-341]) or lysosomal (0.1 µM concanamycin [Conc. A]) inhibition, and in control conditions (DMSO only [Veh.]). Myotube expression levels of muscle specific E3-specific ligase FBXO32 (MuRF1) and TRIM63 (Atrogin-1) (C), and of ubiquitin (UBB) and two components of the proteasome system (UBE2B and PSAM1) were measured by RT-qPCR, normalized against TBP mRNA levels, and expressed as a fold change relative to the SBS condition. Results are the mean ± SEM of at least 3 independent experiments. Significant differences result from paired T-tests (*p < 0.05).
Figure 4
Figure 4
Winter bear serum impacts main factors of autophagy/lysosomal degradation in human muscle cells. Illustrative immunodetection (A) and corresponding quantification (B) of SQSTM1 (p62) and the LC3b II/I ratio in human myotubes exposed for 24 hours to SBS or WBS (C). Myotube expression of CTSL and ATG3 was measured by RT-qPCR, normalized against TBP mRNA levels, and expressed as a fold change relative to the SBS condition (D). Data are the mean ± SEM of at least 3 independent experiments. Significant differences result from paired T-tests (*p < 0.05; **p < 0.01).
Figure 5
Figure 5
Winter bear serum activates mTOR/PKB pathways in human muscle cells. Illustrative blots and corresponding quantification of mTOR, PKB, FOXO3a, S6K, GSK3beta, SGK and their phosphorylated forms. Results are the mean ± SEM of at least of 3 independent experiments. Significant differences result from paired T-tests (*p < 0.05).

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