REGULATORY EFFECTS OF GLUCOSE ON THE CATALYTIC ACTIVITY AND CELLULAR CONTENT OF GLUCOKINASE IN THE PANCREATIC BETA-CELL - STUDY USING CULTURED RAT ISLETS

Glucose regulates the cellular content of glucokinase in the pancreatic beta cell by altering the level of the enzyme. We investigated the existence of a second regulatory pathway, an alteration in the catalytic activity, by comparing V-max and protein levels of glucokinase in rat islets cultured under high glucose conditions (16.7 mM) for 6, 14, and 24 h. The V-max was increased by glucose at all time points. In contrast, glucokinase protein levels on Western blots were unchanged from the control value at 6 h but increased 40% at the later time points (P < 0.0002). Further evidence for a dual regulatory system was obtained with a reversal protocol. After a 6-h incubation at high glucose, an additional 3-h incubation at 5.5 mM glucose restored glucokinase V-max to normal, but failed to change the V-max after a 24-h incubation at high glucose. Finally, 10 mu M cycloheximide partially prevented the increase in glucokinase V-max induced by 24 h of high glucose, but had no effect at 6 h, suggesting the early increase in enzymatic activity did not require protein synthesis. In summary, glucose regulates both the catalytic activity and cellular content of glucokinase in the beta cell. Glucose-induced increases in glucokinase activity are an important element of the beta cell adaptive response to hyperglycemia.