Rabbit Optic Nerve Phosphorylates Glucose through a Glucokinase-like Enzyme: Studies in Normal and Spontaneously Hyperglycemic Animals

Abstract

We investigated glucose phosphorylation at various concentrations of glucose (1, 5, 10, 25, 50, 100 mmol/liter) in rabbit optic nerve. In the 3000g supernatant of whole rabbit optic nerve homogenates from female albino rabbits (n = 10, 1.8-2.0 kg body weight, mean ± SEM morning glycemia: 8.25 ± 0.29 mmol/liter), the glucose phosphorylating activity (NADP reduction measured as change in optical density at 366 nm at pH 7.5) increased progressively with the increase in glucose concentration (r = 0.89; P < 0.05) and approached the maximum at a very high glucose level (100 mmol/liter), with values (mean ± SEM) of 8.75 ± 0.97 nanomol/min/mg protein and 11.57 ± 1.15 at 1 and 100 mmol/liter glucose, respectively (+32.23%; P < 0.01). At a more alkaline pH (8.2;, n = 5, mean ± SEM morning glycemia: 8.83 ± 0.07 mmol/liter) glucose phosphorylation was higher than at pH 7.5 and retained the glucose concentration dependence (r = 0.95, P < 0.01). These kinetic characteristics are reminiscent of those of the low-affinity enzyme glucokinase, which is typically present in the liver. By subtracting the activity at 1 mmol/liter glucose from that at higher glucose concentrations, we calculated the "glucokinase component," which, together with the "hexokinase component," forms the "total" glucose phosphorylating activity. In five rabbits (of similar age and weight) with spontaneous hyperglycemia (mean ± SEM: 11.71 ± 0.60 mmol/liter), the optic nerve glucose phosphorylating activity was lower (value at 1 mmol/liter glucose: 5.42 ± 1.31, −38.06%, P < 0.05). This could contribute to the hyperglycemia by reducing glucose utilization. As in the normal controls, the glucose phosphorylating activity increased progressively (r = 0.98, P < 0.001) with the rise of glucose until 100 mmol/liter, with the value at 100 mmol/liter glucose (8.91 ± 1.41) being increased by +64.39% (P < 0.01) compared to the value at 1 mmol/liter glucose. Thus, in the rabbit optic nerve glucose is phosphorylated through a glucokinase-like enzyme, which entails increased glucose phosphorylation (even if of moderate degree) upon nerve exposure to high-glucose concentrations. In spontaneously hyperglycemic rabbits the actual glucose phosphorylation would depend upon both enzyme level (which is decreased) and glucose concentration (which is increased). Due to the in vivo inhibition of the hexokinase component by glucose-6-phosphate, the glucokinase component may be prevalent, and, therefore, the chronic diabetic hyperglycemia might induce a tendency to increased glucose phosphorylation, which may be relevant to development of optic neuropathy in diabetes.