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Enzymes of glucose metabolism in normal mouse pancreatic islets.

ABSTRACT: 1. Glucose-phosphorylating and glucose 6-phosphatase activities, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP(+)-linked isocitrate dehydrogenase, ;malic' enzyme and pyruvate carboxylase were assayed in homogenates of normal mouse islets. 2. Two glucose-phosphorylating activities were detected; the major activity had K(m) 0.075mm for glucose and was inhibited by glucose 6-phosphate (non-competitive with glucose) and mannoheptulose (competitive with glucose). The other (minor) activity had a high K(m) for glucose (mean value 16mm) and was apparently not inhibited by glucose 6-phosphate. 3. Glucose 6-phosphatase activity was present in amounts comparable with the total glucose-phosphorylating activity, with K(m) 1mm for glucose 6-phosphate. Glucose was an inhibitor and the inhibition showed mixed kinetics. No inhibition of glucose 6-phosphate hydrolysis was observed with mannose, citrate or tolbutamide. The inhibition by glucose was not reversed by mannoheptulose. 4. 6-Phosphogluconate dehydrogenase had K(m) values of 2.5 and 21mum for NADP(+) and 6-phosphogluconate respectively. 5. Glucose 6-phosphate dehydrogenase had K(m) values of 4 and 22mum for NADP(+) and glucose 6-phosphate. The K(m) for glucose 6-phosphate was considerably below the intra-islet concentration of glucose 6-phosphate at physiological extracellular glucose concentrations. The enzyme had no apparent requirement for cations. Of a number of possible modifiers of glucose 6-phosphate dehydrogenase, only NADPH was inhibitory. The inhibition by NADPH was competitive with NADP(+) and apparently mixed with respect to glucose 6-phosphate. 6. NADP(+)-isocitrate dehydrogenase was present but the islet homogenate contained little, if any, ;malic' enzyme. The presence of pyruvate carboxylase was also demonstrated. 7. The results obtained are discussed with reference to glucose phosphorylation and glucose 6-phosphate oxidation in the intact mouse islet, and the possible nature of the beta-cell glucoreceptor mechanism.

SUBMITTER: Ashcroft SJ

PROVIDER: S-EPMC1179311 | biostudies-other | 1970 Aug

REPOSITORIES: biostudies-other

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Project description:1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.

Project description:1. Rates of insulin secretion, glucose utilization, lactate output, incorporation of glucose into glycogen, contents of glucose 6-phosphate, fructose 1,6-diphosphate and ATP, and maximally extractable enzyme activities of hexokinase, high-K(m) glucose-phosphorylating activity (;glucokinase'), glucose 6-phosphatase and unspecific acid phosphatase were measured in isolated pancreatic islets from fed and 48-h-starved mice. 2. In the fed state insulin secretion from isolated islets was increased five- to six-fold when the extracellular glucose concentration was raised from 2.5mm to 16.7mm; 5mm-caffeine potentiated this effect. The secretory response to glucose of islets from mice starved for 48h was diminished at all glucose concentrations from 2.5mm up to approx. 40mm. Very high glucose concentrations (60mm and above) restored the secretory response to that found in the fed state, suggesting that the K(m) value for the overall secretory process had been increased (approx. fourfold) by starvation. Addition of 5mm-caffeine to islets from starved mice also restored the insulin secretory response to 2.5-16.7mm-glucose to normal values. 3. Extractable hexokinase, ;glucokinase', glucose 6-phosphatase and unspecific phosphatase activities were not changed by starvation. 4. Glucose utilization and glycolysis (measured as the rate of formation of (3)H(2)O from [5-(3)H]glucose over a 2h period) was decreased in islets from starved mice at all glucose concentrations up to approx. 55mm. At still higher glucose concentrations up to approx. 100mm, there was no difference between the fed and starved state, suggesting that the K(m) value for the rate-limiting glucose phosphorylation had been increased (approx. twofold) by starvation. Preparation of islets omitting substrates (glucose, pyruvate, fumarate and glutamate) from the medium during collagenase treatment lowered the glucose utilization measured subsequently at 16.7mm-glucose by 38 and 30% in islets from fed and starved mice respectively. Also the 2h lactate output by the islets at 16.7mm extracellular glucose was diminished by starvation. Incorporation of glucose into glycogen was extremely low, but the rate of incorporation was more than doubled by starvation. 5. After incubation for 30min at 16.7mm-glucose the content of glucose 6-phosphate was unchanged by starvation, that of ATP was increased and the concentration of (fructose 1,6-diphosphate plus triose phosphates) was decreased. 6. Possible mechanisms behind the correlated impairment in insulin secretion and islet glucose metabolism during starvation are discussed.

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Enzymes of glucose metabolism in normal mouse pancreatic islets.

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