GLUCOSE MODULATES GLUCOSE TRANSPORTER AFFINITY, GLUCOKINASE ACTIVITY, AND SECRETORY RESPONSE IN RAT PANCREATIC BETA-CELLS

Pancreatic islets were cultured for 24 h in medium containing either low (1.4), normal (5.5), or high (16.7 mM) glucose, and then insulin secretion was measured at the end of 1 h incubation at 37-degrees-C. Insulin release in the absence of glucose was 64 +/- 20, 152 +/- 11, and 284 +/- 30 pg . islet-1 . h-1 (mean SE, n = 6, G1.4 and G16.7 vs. G.5.5, P < 0.05) and the response to 22 mM glucose stimulation was 640 +/- 136, 2460 +/- 276, and 1890 +/- 172 pg . islet-1 . h-1, respectively (n = 6, G1.4 vs. G5.5, P < 0.01, G16.7 vs. G5.5, P = 0.065). The 50% maximal response of insulin secretion (increment over baseline) was reached at an average glucose concentration of 9.9 +/- 0.7 MM in islets preexposed to G5.5, and at glucose 13.3 +/- 0.9 and 4.8 +/- 0.4 mM (P < 0.05 in respect to G5.5) in islets preexposed to G1.4 and G16.7, respectively. To investigate the molecular mechanism responsible for this altered glucose sensitivity, we measured, in parallel experiments, the kinetic characteristics of glucose transport, glucokinase, and glucose utilization. Glucose transport was measured by evaluating 3-O-methylglucose uptake. The apparent K(m) of the low-affinity transporter (GLUT2) was 16.6 +/- 2.4 mM in isolated pancreatic cells cultured at 5.5 mM glucose. In cells cultured at both low (1.4 mM) or high (16.7 mM) glucose concentrations, a significant change in the apparent K(m) of this glucose transporter function was observed (24.4 +/- 2.9 and 7.1 +/- 0.6 mM, n = 5, P < 0.05 and <0.01, respectively), with no change in the V(max) of the uptake. Under the same experimental conditions a concomitant change in glucokinase activity was observed: the enzyme V(max) was 4.9 +/- 0.32, 8.7 +/- 0.79, and 15.8 +/- 0.98 nmol . mug DNA-1 . h-1 in islets exposed to either 1.4, 5.5, or 16.7 mM glucose, respectively (mean +/- SE, n = 5, G1.4 and G16.7 vs. G.5.5, P < 0.05), with no significant change in the enzyme K(m). In control islets glucose utilization, measured by (H2O)-H-3 production from [5-H-3]glucose, had a K(m) of 8.0 +/- 1.7 mM and a V(max) of 8.9 +/- 0.41 nmol . mug DNA-1 . 2 h-1. In islets exposed to either G1.4 or G16.7, the glucose utilization V(max) was decreased and increased (5.3 +/- 0.56 and 13.8 +/- 0.98 nmol - mug DNA-1 .2 h-1, n = 5, P < 0.05), parallel with the changes observed in glucokinase activity. Moreover, the apparent glucose utilization K(m) was increased in G1.4 preexposed islets (15.6 +/- 1.3 mM) and decreased in G16.7 preexposed islets (3.7 +/- 0.9 mM), parallel with the changes observed in the glucose transport K(m). These studies indicate that in vitro the sensitivity and responsiveness of pancreatic islets to glucose may be regulated by the ambient glucose concentrations. They support the concept that glucokinase plays a pivotal role in regulating glucose metabolism V(max), and, therefore, the 13-cell responsiveness to glucose. In addition, they also indicate that changes in the affinity (K(m)) of the glucose transporter are associated to changes in the K(m) of glucose utilization, thus suggesting a possible role of glucose transport in determining the beta-cell sensitivity to glucose.