CONTROL OF GLUCOKINASE TRANSLOCATION IN RAT HEPATOCYTES BY SORBITOL AND THE CYTOSOLIC REDOX STATE

In rat hepatocytes cultured in 5 mM glucose, glucokinase activity is p resent predominantly in a bound state, and during permeabilization of the cells with digitonin in the presence of Mg2+ less than 20% of gluc okinase activity is released. However, incubation of hepatocytes with a higher [glucose] [concn. giving half-maximal activation (A(50)) 15 m M] or with fructose (A(50) 50 mu M) causes translocation of glucokinas e from its Mg2+-dependent binding site to an alternative site [Agius a nd Peak (1993) Biochem. J. 296, 785-796]. A comparison of various subs trates showed that sorbitol (A(50) 8 mu M) was 6-fold more potent than fructose at causing glucokinase translocation, whereas tagatose was a s potent and mannitol was > 10-fold less potent (A(50) 550 mu M). Thes e substrates also stimulate glucose conversion into glycogen with a si milar relative potency, suggesting that conversion of glucose into gly cogen is dependent on the binding and/or location of glucokinase withi n the hepatocyte. Ethanol and glycerol inhibited the effects of fructo se, sorbitol and glucose on glucokinase translocation, whereas dihydro xyacetone had a small additive effect at sub-maximal substrate stimula tion. The converse effects of glycerol and dihydroxyacetone suggest a role for the cytosolic NADH/NAD(+) redox state in controlling glucokin ase translocation. Titrations with three competitive inhibitors of glu cokinase did not provide evidence for involvement of glucokinase flux in glucose-induced glucokinase translocation: N-acetylglucosamine inhi bited glucose conversion into glycogen, but not glucose-induced glucok inase translocation; glucosamine partially suppressed glucose-induced and fructose-induced glucokinase translocation, at concentrations that caused total inhibition of glucose conversion into glycogen; D-mannoh eptulose increased glucokinase release and had an additive effect with glucose. 3,3'-Tetramethylene-glutaric acid (5 mM), an inhibitor of al dose reductase, inhibited glucokinase translocation induced by glucose , but not that by sorbitol or fructose, suggesting that glucose may in duce glucokinase translocation by conversion into sorbitol. Sorbitol g enerated from glucose intrahepatically or extrahepatically in hypergly caemic conditions may be a physiological regulator of hepatic glucokin ase translocation.