Hamiel CR, Pinto S, Hau A, Wischmeyer PE. Glutamine enhances heat shock protein 70 expression via increased hexosamine biosynthetic pathway activity. Am J Physiol Cell Physiol 297: C1509 -C1519, 2009. First published September 23, 2009 doi:10.1152/ajpcell.00240.2009.-Glutamine (GLN) plays a key role in cellular protection following injury via enhancement of heat shock protein 70 (HSP70). The pathway by which GLN enhances HSP70 is unknown. GLN is a key substrate for the hexosamine biosynthetic pathway (HBP), which has been shown to induce HSP70. We sought to explore the role of the HBP in GLN-mediated HSP70 expression. Both chemical inhibitors and small interfering (si)RNA knockdown of key HBP enzymes were used in mouse embryonic fibroblast cells to determine the effects of the HBP on HSP70 expression. The O-glycosylation, nuclear translocation, and transcriptional activation of heat shock factor-1 (HSF-1) and Sp1 were evaluated using immunoprecipitation, Western blotting, and luciferase assays. HSP70 expression levels were evaluated via ELISA and Western blotting. GLN augmented HBP activity before and after heat stress (HS). Chemical inhibition of HBP enzymes reduced GLNmediated HSP70 expression. Specific siRNA targeting of the key HBP enzyme UDP-N-acetylglucosamine (GlcNAc): polypeptide-O--acetylglucosaminyltransferase (OGT) blocked GLN-mediated HSP70 expression and attenuated GLN-mediated cellular protection post-HS. Chemical and siRNA attenuation of the HBP blocked GLN-induced nuclear translocation of Sp1 and HSF-1, which are key to maximal HSP70 expression. Finally, immunoprecipitation revealed HSF-1 was O-glycosylated, and GLN enhanced this effect. These results suggest that metabolism of GLN via the HBP enhances HSP70 expression. This effect appears to be mediated via O-glycosylation, nuclear translocation, and transcriptional activation of Sp1 and HSF-1. This is an important mechanistic description of a pathway that appears responsible for GLNmediated HSP70 expression.heat shock proteins; O-GlcNAc; O-glycosylation; heat shock factor-1; cell protection; cell survival; cell injury HEAT SHOCK PROTEINS (HSPs) are highly conserved proteins involved in the most basic mechanisms of cellular protection. HSP expression following a sublethal insult can induce "stress tolerance" and provide protection from subsequent stress that would otherwise be lethal (8,13,19,20). Previous data indicate that the induction of HSPs (particularly HSP70) can provide significant protection against many forms of injury (8,13,19,20). However, these findings have yet to be applied in a clinical setting because common laboratory inducers of HSPs are not safe for human administration. Our laboratory has shown that glutamine (GLN) can safely enhance HSP expression in in vitro and in vivo settings (9,16,18,23,24). Using genetic knockout models, we have also established that HSP induction is necessary for GLN's beneficial effect following injury (9, 18). Animals and cells that lack the HSP70 gene or the heat shock factor-1 (HSF-1) gene sho...