EVIDENCE FOR A ROLE OF GLUCOSE-INDUCED TRANSLOCATION OF GLUCOKINASE IN THE CONTROL OF HEPATIC GLYCOGEN-SYNTHESIS

Glucokinase reversibly partitions between a bound and a free state in the hepatocyte in response to the metabolic status of the cell. Maximu m binding occurs at low [glucose] (<5 mM) and minimum binding at high [glucose] or in the presence of sorbitol or fructose. In this study we determined the binding characteristics of glucokinase in the hepatocy te in situ, by adenovirus-mediated glucokinase overexpression combined with the digitonin-permeabilization technique. We also determined the sensitivity of glycogen synthesis to changes in either total glucokin ase overexpression or in free glucokinase activity. Glucokinase overex pression is associated with an increase in both free and bound activit y, with an overall decrease in the proportion of bound activity, In he patocytes incubated at low [glucose] (0-5 mM), glucokinase binding inv olves a high-affinity binding site with a K-d of similar to 0.1 mu M a nd a binding capacity of similar to 3 pmol/mg total cell protein and l ow-affinity binding with a K-d of similar to 1.6 mu M. Increasing gluc ose concentration to 20 mM causes a dose-dependent increase in the K-d of the high-affinity site to similar to 0.6 mu M, and this effect was mimicked by 50 mu M sorbitol, a precursor of fructose 1-P, confirming that this site is the regulatory protein of glucokinase. Glycogen syn thesis determined from the incorporation of [2-H-3,U-C-14]glucose into glycogen at 5 man or 10 mM glucose was very sensitive to small increa ses in total glucokinase activity and correlated more closely with the increase in free glucokinase activity. The relation between glycogeni c flux and glucokinase activity is sigmoidal. Expression of the sensit ivity of glycogen synthesis to glucokinase activity as the control coe fficient reveals that the coefficient is greater for the incorporation of 2-tritium (which occurs exclusively by the direct pathway) than fo r incorporation of C-14 label (which involves direct and indirect path ways) and is greater at 5 mM glucose (when glucokinase is maximally se questered at its high-affinity site) than at 10 mM glucose. The result s support the hypothesis that compartmentation of glucokinase in the h epatocyte increases the sensitivity of glycogen synthesis to small cha nges in total glucokinase activity and that glucose-induced translocat ion of glucokinase has a major role in the acute control of glycogen s ynthesis.