Glucosamine-induced phosphorylation of the at-subunit of eukaryotic initiation factor 2 is mediated by the protein kinase R-like endoplasmic-reticulum associated kinase

In diabetic animals, enhanced production of vascular endothelial growth factor is thought to be a major contributor to the development of diabetic retinopathy. In the present study, glucosamine-treated R28 retinal neuronal cells were used as an experimental model system to explore the possible involvement of the hexosamine biosynthetic pathway in the diabetes-induced changes in mRNA translation. Glucosamine treatment enhanced vascular enclothelial growth factor production subsequent to changes in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2, with no change in vascular endothelial growth factor mRNA content. Possible mechanisms through which glucosamine might act to increase eukaryotic initiation factor 2 alpha phosphorylation include enhanced O-linked glycosylation of protein kinase or phosphatase regulatory proteins and/or induction of oxidative stress. However, increasing global protein O-glycosylation through inhibition of O-beta-N-acetylglucosaminidase did not mimic the effect of glucosamine on eukaryotic initiation factor 2 alpha phosphorylation. Likewise, attenuating glucosamine-induced oxidative stress with two different antioxidants did not reduce glucosamine-induced eukaryotic initiation factor 2 alpha phosphorylation. Glucosamine treatment was also found to promote eukaryotic initiation factor 2 alpha phosphorylation in wild-type mouse embryonic fibroblasts, but not in mouse embryonic fibroblasts lacking the eukaryotic initiation factor 2a kinase referred to as RNA-dependent protein kinase-like endoplasmic-reticulum associated kinase, implicating the kinase in the glucosamine-induced increase in eukaryotic initiation factor 2 alpha phosphorylation. Overall, the results are consistent with glucosamine causing activation of RNA-dependent protein kinase-like endoplasmic-reticulum associated kinase, which phosphorylates eukaryotic initiation factor 2 alpha and consequently upregulates translation of mRNAs encoding specific proteins, such as vascular endothelial growth factor. (c) 2005 Elsevier Ltd. All rights reserved.