Dagmar Zahner

Hexose metabolism in pancreatic islets stimulation by d-glucose of [2-3H]glycerol detritiation

1. In pancreatic islets, a rise in glucose concentration is known to increase the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization. The opposite situation was found to prevail in parotid cells. 2. In rat pancreatic islets, D-glucose caused a concentration-related stimulation of 3H2O production from [2-3H]glycerol, but failed to affect 3H2O production from [1(3)-3H]glycerol or 14CO2 production from [U-14C]glycerol. At the low concentration used in most of these experiments (i.e. 1.0 mM), glycerol failed to affect D-[U-14C]glucose oxidation. 3. These findings suggest that the preferential stimulation by D-glucose of mitochondrial oxidative events in pancreatic islets represents an unusual situation in secretory cells and involves an accelerated circulation in the glycerol phosphate shuttle.

Enzyme-to-enzyme tunnelling between phosphoglucoisomerase and phosphofructokinase

1. Cross-linked and permeabilized rat erythrocytes were incubated for 2-5 min at 37 degrees C in the presence of ATP and either D-[U-14C]glucose 6-phosphate (3 mM) mixed with unlabelled D-fructose 6-phosphate (1 mM) or D-[U-14C]fructose 6-phosphate (1 mM) mixed with unlabelled D-glucose 6-phosphate (3 mM). 2. The contribution of molecules derived from the radioactive ketohexose ester relative to the total amount of newly formed D-fructose 1,6-bisphosphate was lower than the time-related average value for such a relative contribution in the pool of D-fructose 6-phosphate. 3. From such a difference, it was calculated that, under the present experimental conditions, 13.1 +/- 2.0% of the molecules of D-fructose 1,6-bisphosphate formed during incubation are directly derived from D-glucose 6-phosphate by a process of enzyme-to-enzyme channelling between phosphoglucoisomerase and phosphofructokinase, rather than originating from the free pool of D-fructose 6-phosphate. 4. A comparable value of 13.2 +/- 3.2% was reached when the process of enzyme-to-enzyme tunnelling was judged from the 3H/14C ratio in D-fructose 1,6-bisphosphate formed by permeabilized erythrocytes exposed for 5-15 min to D-glucose 6-phosphate (3 or 5 mM) mixed with tracer amounts of both D-[1-14C]glucose 6-phosphate and D-[2-3H]glucose 6-phosphate.

Interconversion of D-fructose 1,6-bisphosphate and triose phosphates in human erythrocytes

Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.

Anomeric specificity of liver glycogenolysis

In rat liver slices incubated in the absence of exogenous D-glucose, both the basal and glucagon-stimulated output of D-glucose resulted in the production of a greater relative amount of alpha-D-glucose than that found at anomeric equilibrium. Comparable results were obtained in isolated hepatocytes. In these experiments, the rate of glycogenolysis largely exceeded that of glycogen synthesis. These findings indicate that liver glycogenolysis represents an alpha-stereospecific process.

Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level

A radioisotopic method for the assay of reduced or oxidized pyridine nucleotides, based on the interconversion of 2-[U-14C]ketoglutarate or 2-keto[3,4-3H]glutarate and labelled L-glutamate in the reaction catalyzed by glutamate dehydrogenase, was applied to the measurement of lactate dehydrogenase activity in rat pancreatic islet homogenates. Using the tritiated tracer, the limit of sensitivity of the procedure for NAD(P)H assay was close to 1.0 fmol/sample, and lactate dehydrogenase activity could be measured in as little as 0.0005 islet/sample i.e., at a single cell level. This radioisotopic procedure, which can be used for the assay of various metabolites and enzymic activities, thus provides a tool for investigating the heterogeneity in metabolic behaviour of individual cells.

NADP-malate dehydrogenase activity in rat erythrocytes. Comparison with pyruvate kinase in relation to coupling with lactate dehydrogenase

The present study explores the possible channelling of pyruvate generated by either pyruvate kinase or NADP-malate dehydrogenase to lactate dehydrogenase in cross-linked and permeabilized erythrocytes. The generation of both unlabelled and 14C-labelled pyruvate and lactate was measured in rat erythrocytes, which were prepared for cross-linking with dimethyl suberimidate and permeabilization by digitonin and then exposed to unlabelled or 14C-labelled malate and/or phospho-enol-pyruvate. Rat erythrocytes were found to display NADP-malate dehydrogenase activity. Under conditions in which the generation rates of pyruvate from either phospho-enol-pyruvate (15 microM) or malate (0.5 mM) were not vastly different from one another, a greater fraction of the 2-keto acid was converted to lactate when produced from phospho-enol-[1-14C]pyruvate rather than [U-14C]malate. This difference was most obvious when the availability of exogenous NADH was close to or somewhat below that theoretically required to ensure full conversion of endogenously formed pyruvate to lactate. These findings are compatible with the view that pyruvate generated at the pyruvate kinase level is converted to lactate more efficiently than pyruvate produced in the reaction catalysed by NADP-malate dehydrogenase.

Kinetic behaviour of liver glucokinase in insulinopenic situations: effect of fructose 1-phosphate in fed and starved rats

In the liver postmicrosomal supernatant of starved rats, the high-Km glucose-phosphorylating enzymic activity, presumably attributable to glucokinase, is not solely decreased but also displays an apparently lower affinity and less pronounced temperature dependency than in fed rats. In the presence of exogenous D-glucose 6-phosphate and D-fructose 6-phosphate, the rate of D-glucose phosphorylation is enhanced by D-fructose 1-phosphate at low (10 mM) but not high (90 mM) hexose concentration and at high (30-37 degrees C) but not low (10 degrees C) temperature. The responsiveness of glucokinase to D-fructose 1-phosphate is apparently decreased in starved animals. Nevertheless, the latter ester does not suppress the starvation-induced alteration in both the apparent affinity and temperature dependency of liver glucokinase. It is proposed, therefore, that such an alteration may reflect changes in the intrinsic properties of the high-Km hepatic enzyme.