title: Diabetic levels of glucose increase cellular reducing equivalents but reduce survival in three models of 661W photoreceptor-like cell injury.
abstract: BACKGROUND
  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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  661W 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.
  
  
  CONCLUSIONS
  Acute 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.
authors.affiliation: Ophthalmology Research Unit, Gallipoli Medical Research Institute, Newdegate St, Greenslopes, Brisbane, 4120, Australia. c.layton@uq.edu.au.|School of Medicine, University of Queensland, Herston Rd, Herston, Brisbane, 4006, Australia. c.layton@uq.edu.au.|Ophthalmology Department, Greenslopes Hospital, Newdegate St, Greenslopes, Brisbane, Australia. c.layton@uq.edu.au.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Layton
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000633:toxicity; D005434:Flow Cytometry; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D008954:Models, Biological; D009249:NADP / Q000378:metabolism; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D010786:Photoreceptor Cells / Q000187:drug effects* / Q000473:pathology* / Q000528:radiation effects; D011833:Radiation Injuries, Experimental / Q000209:etiology; D019311:Staurosporine / Q000633:toxicity
chemical_list: D017304:Annexin A5; D004791:Enzyme Inhibitors; D016877:Oxidants; D009249:NADP; D006861:Hydrogen Peroxide; D053148:Caspase 3; D053179:Caspase 7; D019311:Staurosporine; D005947:Glucose