We evaluated the possible role of islet glucokinase in controlling the rate of islet glucose metabolism, and thereby the rate of glucose-induced insulin release. The activities of glucokinase, hexokinase, P-fructokinase, and glyceraldehyde-P dehydrogenase were quantitated in sonicated or isotonically homogenized islet preparations using pyridine nucleotide-dependent fluorometric assays. In sonicates, about 1/4 of the islet glucose phosphorylating activity was due to an enzyme with kinetic properties similar to glucokinase; 3/4 of the activity was due to hexokinase. The procedure for determining islet glucokinase activity was improved by centrifuging isotonic islet homogenates at 12,000 x g. The supernatant fraction was enriched for glucokinase. About 1/2 of the glucose phosphorylating activity in this fraction was due to glucokinase and 1/2 was due to hexokinase. The glucokinase activity in islet homogenates was !23 of the activity of hexokinase, 1/40 of the activity of P-fructokinase, and 1/400 of the activity of glyceraldehyde-P dehydrogenase. Detailed concentration dependency curves of glucose and mannose utilization were also obtained with intact isolated pancreatic rat islets. Glucose and mannose usage in islets was governed by two superimposed hyperbolic systems differing in Km and Vmax. A high Km system (Km for glucose 11 mM and for mannose 21 mM) predominated. A low Km system (Km for glucose 215 and for mannose 530 microM) contributed about 15% to the total activity. The available data with intact islets could be rationalized by the existence of two distinct hexose phosphorylating enzymes with differing capacities and kinetic properties. These enzymes, tentatively identified as glucokinase and hexokinase, could coexist in the same cell or could be distributed among different cell types. The possible physiologic significance of these results is discussed, emphasizing the idea of dual control of glycolysis and insulin release by glucokinase and hexokinase. An earlier proposal that glucokinase serves as glucoreceptor of beta-cells [J. Biol. Chem. 243:2730 (1968)] is greatly strengthened by the present studies.

Starvation refeeding experiments were conducted in rats to test the hypothesis that adaptation of glucokinase (the high Km component of glucose phosphorylation) could be the major determinant of glucose metabolism of pancreatic islet cells and of glucose-stimulated insulin release. It was found that glucokinase of islet homogenates, glucose use by intact isolated islets, and glucose-induced insulin release as studied in a perifusion system were decreased after 24 h of fasting, whereas P-fructokinase and 3-P-glyceraldehyde DH were unaltered. After extended fasting (e.g., 120 h) all three enzymes were decreased but glucose use did not change any further. Refeeding normalized all parameters. These and previous results support the concept that glucokinase serves as the adaptive beta-cell glucoreceptor relating blood glucose to insulin release.

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We examined the relationship between glucose-induced insulin release and the intermediary metabolism of islets from fed and fasted rats. Isolated islets were perifused and insulin release measured in the effluent. At various times after switching islets from 2.4 to 8.6 or 14.5 mM glucose or from 2.4 to 14.5 and back to 2.4 mM glucose, islets were quickly frozen, freeze dried, and subsequently analyzed for tissue content of glucose-6-P, fructose-1,6-P2 plus triose-P, Pi, ATP, ADP, 5'-AMP, NADH, NADPH, total NAD, and total NADP using enzymatic fluorometric procedures. When islets from fed rats were exposed to high glucose, there were concomitant increases of insulin release and islet content of glucose-6-P, fructose-1,6-P2 plus triose-P, NADH, and NADPH. During stimulation Pi and 5'-AMP content fell markedly. The total adenine nucleotide content remained constant. Similar secretory and metabolic changes occurred when 1.5 mM Pi was added to the perifusion fluid. When glucose-stimulated islets were switched back to low glucose for 10 min, all substances but fructose-1,6-P2 plus triose-P, 5'-AMP, NADPH, and possibly ATP returned to the prestimulatory level. Starvation of rats for 3 days blocked the secretory response to 8.6 mM glucose. Fructose-1,6-P2 plus triose-P rose but it did not attain the level existing in islets from fed rats. The ratios (ATP)/(5'-AMP) and (ATP)/(Pi)(adp) increased to the values observed in glucose-stimulated islets of fed rats. The metabolic changes in islets from fed rats exposed to high glucose are consistent with an activation of glycolysis occurring concomitantly with stimulated rates of insulin release. This occurs despite the decrease of important activators of glycolysis--Pi and 5'-AMP. The enhanced glycolysis possibly results from P-fructokinase activation by increased fructose-6-P levels. Activation of glycolysis with 8.6 mM glucose was not as pronounced in islets from starved rats. Despite the different secretory response of islets from fet and fasted rats, the changes of phosphorylation state in the islets, in particular, Pi and 5'-AMP levels, were similar.