MEASUREMENT AND MODELING OF GLUCOSE-6-PHOSPHATASE IN PANCREATIC-ISLETS

In the beta-cells of the pancreas, glucose phosphorylation carried out by glucokinase is the rate-controlling step in glycolysis, and the ki netic characteristics of glucokinase govern to a high degree the dose- response relationship between glucose and insulin release. Because glu cose-6-phosphatase (G-6-Pase) opposes the action of glucokinase, it ma y have a regulatory role in the release of insulin in response to gluc ose if the enzyme is present in the beta-cells. A number of researcher s have reported finding high levels of G-6-Pase in islets, but quantit ation of its activity remains controversial, mainly because of difficu lties in solubilizing a particulate enzyme. Therefore a method develop ed to measure functional glucose phosphorylation activity in intact br ain was applied (Chi, M. M.-Y., M. E. Pusateri, J. G. Carter, B. J. No rris, D. B. McDougal, Jr., and O. H. Lowry. Anal. Biochem. 161: 508-51 3, 1987), and the rates of accumulation and disappearance of 2-deoxygl ucose 6-phosphate (DG-6-P) in freshly harvested islets were determined as a measure of glucose cycling. Islets were incubated in the presenc e of 30 mM 2-deoxyglucose (DG) for 60 min, and subsequently the incuba tion medium was replaced with medium containing no DG, but instead hig h levels of mannoheptulose as a blocker of phosphorylation. The conten t of DG-6-P in the islets was measured at strategic times during the p rotocol. As predicted by a mathematical model, DG-6-P accumulated in t he presence of DG and decayed after its washout. Both of these results are consistent with islets containing dephosphorylation activity for this substrate. The kinetic curves were fit using a mathematical model , and the maximal G-6-Pase activity was estimated to be 0.13 +/- 0.005 mu mol . g(-1). min(-1). However, when the physiological effect of th is amount of G-6-Pase activity was assessed by use of a model of glyco lysis, it was found that the impact on glucose cycling and usage was i nsignificant. It was concluded that normal islets do contain measurabl e activity for dephosphorylating glucose 6-phosphate but that this enz ymatic reaction does not play a role in glucose metabolism and sensing by the normal beta-cell.