Glucose Modulates Glucose Transporter Affinity, Glucokinase Activity, and Secretory Response in Rat Pancreatic β-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°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 μmol · μg DNA⁻¹ · h⁻¹ in islets From the Institute of Internal Medicine, Metabolism and Endocrinologyexposed 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 3H₂0 production from [5-³H]glucose, had a K_m of 8.0 ± 1.7 mM and a V_max of 8.9 ± 0.41 nmol · μg 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 · μg 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 β-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 β-cell sensitivity to glucose.