Abstract
BackgroundThe effect of excess glucose on retinal cellular health remains controversial, and cellular reducing equivalents, as indicators of cellular energy production, are widely used as substitute indicators of retinal cellular health. These investigations hypothesised that excess energy substrate availability, as occurs in the diabetic retina, increases the susceptibility of retinal neurons to injury in the presence of increased cellular reducing equivalents.MethodsThe response of 661W cells to phototoxicity, oxidative stress induced by H2O2 and apoptosis induction by staurosporine was characterised in the presence of 5mM glucose and B27 defined media without insulin. Cellular insult was produced by phototoxicity, H2O2 and the apoptosis induction agent staurosporine. The effect of physiologically relevant alterations in environmental glucose on cellular reducing equivalents was assessed by MTT dye reduction and NAD(P)H assays, and cell survival was assessed via caspase 3/7 activation and Annexin V/PI flow cytometry.Results661W photoreceptor-like cells underwent dose dependent cell death primarily by apoptosis in response to phototoxic insult, H2O2, and staurosporine by all measures of cellular viability. Exposure of cells to 25mM glucose (diabetic-type conditions) increased cell death in response to all insults as measured by caspase 3/7 activation and Annexin V/PI flow cytometry. Cellular reducing equivalents were nonetheless increased in all models of injury in the presence of excess glucose. The mechanism of this increase was partly due to increased NADPH but not NADH levels in the presence of 25mM glucose.ConclusionsAcute exposure to 25mM glucose decreased the resilience of 661W photoreceptor-like cells to a range of cellular stressors whilst maintaining or increasing cellular reducing equivalents, partly be increasing NADPH levels. This shows that in 661W cells, diabetic levels of glucose decrease cellular resilience to injury. The decoupling of cellular reducing equivalents levels from cell survival has important implications when investigating the mechanisms of neuronal damage in diabetic retinal neuropathy.
Highlights
The effect of excess glucose on retinal cellular health remains controversial, and cellular reducing equivalents, as indicators of cellular energy production, are widely used as substitute indicators of retinal cellular health
As the changes associated with diabetic retinal neuropathy manifest early in diabetes, an understanding of the mechanisms of retinal neuronal damage in the disease demands that the effect of high environmental glucose on the resilience of retinal neuronal cells is explored
Glucose is a fundamental source of energy for retinal cells [4], and it seems intuitive that the presence of abundant glucose will assist retinal neuronal survival and function, many studies have suggested that excess mitochondrial free radical production in response to higher flux through the electron transport chain in conditions of excess energy substrate is the cause of diabetic complications [5, 6]
Summary
The effect of excess glucose on retinal cellular health remains controversial, and cellular reducing equivalents, as indicators of cellular energy production, are widely used as substitute indicators of retinal cellular health These investigations hypothesised that excess energy substrate availability, as occurs in the diabetic retina, increases the susceptibility of retinal neurons to injury in the presence of increased cellular reducing equivalents. Diabetic retinopathy is becoming increasingly recognised as a panretinal disease beginning early in diabetes with alterations in neuronal function (“diabetic retinal neuropathy”) prior to the onset of a diabetic retinal vasculopathy [1, 2]. This vasculopathy is eventually observable ophthalmoscopically and is the cause of clinical vision loss [3]. Whether glucose promotes survival or cell death in retinal cells remain controversial: a protective effect of glucose on stressed retinal cells has been confirmed in conditions of ischaemia [7], respiratory chain inhibition [8] and glaucoma [9], studies in primary retinal culture show glucose inhibits the protective effect of neurotrophic factors [10], and other CNS neurons have been shown to be less resilient to insult in the presence of high environmental glucose [11]
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