Glucose Modulates Glucose Transporter Affinity, Glucokinase Activity, and Secretory Response in Rat Pancreatic β-Cells
Authors: Purrello Francesco , Buscema Massimo , M Rabuazzo Agata , Caltabiano Venera , Forte Fiorella , Vinci Carmela , Vetri Mario , Vigneri Riccardo 
Published: September 2013

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 &lt; 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 &lt; 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 &lt; 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 Km 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 Km of this glucose transporter function was observed (24.4 ± 2.9 and 7.1 ± 0.6 mM, n = 5, P &lt; 0.05 and &lt; 0.01, respectively), with no change in the Vmax of the uptake. Under the same experimental conditions a concomitant change in glucokinase activity was observed: the enzyme Vmax 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 &lt; 0.05), with no significant change in the enzyme Km. In control islets glucose utilization, measured by 3H20 production from [5-3H]glucose, had a Km of 8.0 ± 1.7 mM and a Vmax 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 Vmax was decreased and increased (5.3 ± 0.56 and 13.8 ± 0.98 nmol · μg DNA−1 · 2 h−1 n = 5, P &lt; 0.05), parallel with the changes observed in glucokinase activity. Moreover, the apparent glucose utilization Km 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 Km. 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 Vmax, and, therefore, the β-cell responsiveness to glucose. In addition, they also indicate that changes in the affinity (Km) of the glucose transporter are associated to changes in the Km of glucose utilization, thus suggesting a possible role of glucose transport in determining the β-cell sensitivity to glucose.