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L-Threonine treatment enhances heat shock protein 25 expression and prevents apoptosis in heat-stressed intestinal epithelial-18 cells

Introduction

Osmotically acting amino acids, such as glutamine, can be cytoprotective following injury in vitro and in vivo. As threonine (THR) can also induce cell-swelling, the aim of this study was to investigate the potential for THR to induce cellular protection in intestinal epithelial-18 cells and to elucidate the mechanism by which it may work.

Methods

Cells were treated for 15 minutes with increasing doses of THR up to 20 mM, with/without subsequent heat stress (HS) injury. Cell survival was evaluated via MTS assay 24 hours following lethal HS (44°C × 50 min). All HS groups were normalized to their non-HS controls. Western blot analysis was used to determine active caspase-3 activity (an indicator of apoptosis), and heat shock protein 25 (HSP25) expression/cellular localization in cells subjected to non-lethal HS (43°C × 45 min). Enhanced nuclear translocation of HSP25 has been linked to decreased apoptosis. Microscopy was used to visualize cell size and morphology since cytoplasmic HSP25 has been shown to enhance actin stabilization during cellular stress.

Results

THR increased cell survival in a dose-dependent manner (P = 0.008 vs. HS controls (CT)), n = 3. A control amino acid cocktail (20 mM valine, alanine and phenylalanine) failed to provide protection from lethal heat stress. Active caspase-3 activity was highest in HS cells and decreased with THR (P = 0.0006 vs. HS CT). HSP25 was predominantly cytoplasmic in non-HS cells and increased in a dose-dependent manner with THR (P = 0.0006 vs. CT). HS caused nuclear translocation of HSP25, and this effect was increased even further with THR treatment (P < 0.05 vs. HS CT). Microscopy showed preserved cell size and structural integrity of the actin cytoskeleton in HS cells treated with THR. Cell size decreased during HS by 40 ± 5% (P = 0.003 vs. non-HS controls). These effects were completely attenuated with THR treatment (P = 0.00001 vs. HS CT).

Conclusion

THR protected cells from lethal HS by decreasing apoptosis. THR can induce HSP25 in CT cells, and enhance nuclear translocation in HS cells. THR treatment preserved the structural integrity of the actin cytoskeleton and prevented cellular crenation during HS. It is possible that THR's mechanism of cellular protection involves cytoskeletal stabilization and decreased apoptosis-mediated by HSP25.

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Hamiel, C., Kallweit, A., Beck, R. et al. L-Threonine treatment enhances heat shock protein 25 expression and prevents apoptosis in heat-stressed intestinal epithelial-18 cells. Crit Care 13, P372 (2009). https://doi.org/10.1186/cc7536

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Keywords

  • Heat Stress
  • Nuclear Translocation
  • Cellular Protection
  • Increase Cell Survival
  • Control Amino Acid