Excessive Hexosamines Block the Neuroprotective Effect of Insulin and Induce Apoptosis in Retinal Neurons

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mM glucose for 24 h attenuated the ability of 10 nM insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mM glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased ∼2- and 15-fold after a 24-h incubation in 20 mM glucose and 1.5 mM glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mM glucose, but not that of 1.5 mM glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, nglucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.