• Top
• Introduction
• Methods
• Results
• Conclusion

Introduction

The purpose of this study was to determine whether glutamine (GLN)-mediated cellular protection is dependent on the O-linked-N-acetylglucosamine (O-glcNAc) pathway. GLN can protect against critical illness via induction of HSP70. The molecular mechanism by which GLN enhances HSP70 is unknown. GLN can increase flux through the hexosamine biosynthetic pathway and activate transcription factors by O-glcNAc. We investigated GLN's effect on O-glcNAc levels and nuclear translocation of SP1 and HSF-1, which are vital to HSP70 expression. To determine the importance of O-glcNAc, we used silencing RNA (siRNA) against O-linked-N-acetylglucosamine transferase (OGT), the enzyme that catalyzes addition of O-glcNAc to proteins.

Methods

Mouse embryonic fibroblast cells were treated with 0 mM GLN (CT) or 10 mM GLN (GLN), heat stressed (HS) and allowed to recover for 20 minutes. Cells were stained and mean fluorescent intensities (MFIs) measured for total O-glcNAc and nuclear HSF-1 and SP1. For OGT silencing, cells were transfected with either no siRNA, siRNA to OGT, or negative control oligos (nc siRNA) and then treated as above (but with 4 hours recovery). HSP70 and OGT were evaluated by western blot.

Results

Microscopy showed GLN treatment increased nuclear MFI for HSF-1 by 40% (HS-CT: 1,005 ± 146 vs HS-GLN:1403 ± 102, P < 0.05) and SP1 by 54% (HS-CT: 214 ± 14 vs HS-GLN: 330 ± 13, P < 0.05). Total O-glcNAc levels showed 44% MFI increase in HS-GLN compared with HS-CT (HS-CT: 360 ± 24 vs HS-GLN: 518 ± 51, P < 0.05). Following OGT silencing, HS-GLN showed a threefold increase in HSP70 (P = 0.04). These increases were completely blocked by OGT silencing (P = 0.02 vs non-siRNA GLN groups). GLN-nc siRNA groups did not decrease in HSP70 production. OGT was knocked down 86% compared with controls (siRNA: 0.999 ± 0.19 vs CT: 0.131 ± 0.05). N = 3.

Conclusion

These results show GLN can activate the O-glcNAc pathway and enhance nuclear translocation of HSF-1 and SP1. Inhibition of OGT blocked GLN-mediated induction of HSP70. Thus, it appears the mechanism of GLN-mediated HSP70 expression is dependent on enhanced O-glcNAc pathway activation.

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