Possible role of free radicals generated by pseudohypoxia in the regulation of hepatic glucose output. An in vitro model using rat liver microsomal glucose 6-phosphatase.

Hepatic glucose output is decreased by hyperglycaemia through an unknown mechanism. We hypothesize that free radicals generated by hyperglycaemic pseudohypoxia might cause glucose output to decrease by inhibiting glucose 6-phosphatase - a key enzyme of gluconeogenesis. To prove this a model experiment was performed on a microsome fraction of rat liver. One of the characteristic features of pseudohypoxia due to hyperglycaemia is an increase in the ratio of NADH/NAD+, so in the present study the changes in NADH - induced glucose 6-phosphatase activity were investigated as related to the release of inorganic phosphate (Pi) derived from glucose 6-phosphate. After incubation for 50 min, Pi release was significantly reduced by NADH (4.026 +/- 0.189 vs 2.696 +/- 0.429 micromol x l(-1) x mg protein(-1), control vs NADH samples, p < 0.01). The decrease in the activity of glucose 6-phosphatase generated by NADH was prevented by using desferrioxamine, an irreversible ferric chelator, butylated hydroxytoluene and Trolox, two agents which inhibit lipid peroxidation, and reduced glutathione, a non-specific radical scavenger. Superoxide dismutase, catalase and the hydroxyl radical scavenger dimethyl sulphoxide proved to be ineffective. When the above investigations were carried out in the presence of a ferric-EDTA complex the inhibition of glucose 6-phosphatase was found to be inducible by hydrogen peroxide and/or hydroxyl free radicals. These investigations seem to indicate that pseudohypoxia due to hyperglycaemia can inhibit the activity of glucose 6-phosphatase both by lipid peroxidation and by inducing hydrogen peroxide and/or hydroxyl free radicals and thus it may play a part in the glucose-induced decrease of hepatic glucose output.