MODULATION OF GLUCOSE RESPONSIVENESS OF INSULINOMA BETA-CELLS BY GRADED OVEREXPRESSION OF GLUCOKINASE

Insulinoma beta-cells capable of overexpressing glucokinase under the control of a doxycycline-dependent transcriptional transactivator were established from parental INS-1 cells. Glucokinase could be maximally induced to a level more than 20 times the basal level after 36 h of c ulture with doxycycline. Intermediate levels of induction could be ach ieved by varying doses of, and time of culture with, the inducer. The rate of glycolysis was measured in cells with 3-, 5-, and 8-fold incre ment in glucokinase activity above the noninduced level. Proportionate increases in glycolytic flux occurred in cells cultured at low physio logical glucose concentration, At high glucose concentration, inductio n of glucokinase in excess of 2-fold above basal resulted in little ad ditional increase in glycolysis. The consequences of graded increases of glucokinase on two physiological glucose effects were investigated, Increments in glucokinase activity were accompanied by a stepwise shi ft to the left of the dose-response curve for the inductive effect of glucose on the L-type pyruvate kinase mRNA. Similarly, the insulin sec retory response to glucose was shifted leftward in glucokinase-induced cells. The following conclusions are drawn: (i) glucokinase is the ma jor rate-limiting enzyme for glycolysis in these cells; (ii) downstrea m metabolic steps become limiting at high extracellular glucose concen tration with moderate increases in glucokinase over the wild-type leve l; (iii) within limits, glucokinase activity is a determining factor f or two types of glucose responses of the beta-cen, the induction of sp ecific gene expression, and insulin release.