The primary aim of these experiments was to assess in vitro effects of hyperglycemia (30 mmol/l glucose) and hypoxia (PO 2 ‫؍‬ 36 torr) of 2-h duration, separately and in combination, on cytosolic and mitochondrial free NADH (NADHc and NADHm, respectively) in retinas from normal rats. NADH is the major carrier of electrons and protons that fuel ATP synthesis and several metabolic pathways linked to diabetic complications. T he importance of duration and severity of hyperglycemia in the development of diabetic retinopathy was clearly established by the Diabetes Control and Complications Trial (1); however, the mechanisms that mediate early and late retinal vascular dysfunction and structural changes remain unclear.Proliferative retinopathy is widely attributed to increased production of vascular endothelial growth factor evoked by hypoxia/ischemia caused by capillary closure and nonperfusion that develop relatively early after the onset of diabetes (2).The possibility that hyperglycemia and hypoxia may interact via a common metabolic imbalance(s) to initiate and/or exacerbate complications of diabetes is suggested by the correspondence of several redox, metabolic, and pathophysiological changes evoked by either condition alone. Examples include an increased ratio of reduced to oxidized free cytosolic NADc (3-16), increased production of free radicals (3,17-24) and vascular endothelial growth factor (17,19,(25)(26)(27)(28), accumulation of triose phosphates (7,9,10,13-15), activation of protein kinase C (PKC) (22,28 -30), decreased Na ϩ /K ϩ -ATPase activity (3,30 -33), early increases in blood flow (3,(33)(34)(35), and paradoxical protective effects of diabetes and brief periods of hypoxia/ ischemia (i.e., ischemic preconditioning) that attenuate dysfunction/injury evoked by subsequent more severe hypoxia/ischemia (3,29,36 -38).An increase in free NADH/NAD ϩ c appears to be the best candidate metabolic imbalance for mediating pathophysiological changes common to diabetes and hypoxia (3,38). This redox imbalance develops when electrons and protons are transferred to free oxidized NAD ϩ c (reducing it to NADHc) faster than electrons and protons carried by NADHc are used for ATP synthesis in mitochondria by oxidative phosphorylation. Hypoxia increases free NADHc because lack of O 2 impairs utilization of electrons and protons carried by mitochondrial NADHm for oxidative phosphorylation. The mass action effect of electrons and protons accumulating in NADHm restrains transfer of electrons and protons from free NADHc to NAD ϩ m by the malate-aspartate electron shuttle; thus, electrons and protons accumulate in, and elevate, free NADHc.In cells for which glucose uptake is insulin-independent, hyperglycemia augments glucose uptake and metabolism via the sorbitol pathway. In the second step of the pathway, sorbitol is oxidized to fructose by sorbitol dehydrogenase, which catalyzes transfer of a hydride ion (:H Ϫ ) from sorbitol to free NAD ϩ c (reducing it to NADHc) and removal of a second hydrogen atom that is released i...