Metabolism of D-glucose anomers in rat pancreatic islets exposed to equilibrated D-glucose.

This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.     

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.     

Enzyme-to-enzyme channelling in the early steps of glycolysis in rat pancreatic islets

The metabolism of D-glucose displays anomeric specificity in rat pancreatic islets. The aim of the present report is to investigate whether such a situation implies enzyme-to-enzyme tunnelling of metabolites in the early steps of glycolysis. For such a purpose, the modelling of alpha- and beta-D-glucose catabolism, itself based on available information concerning both the utilisation of these two anomers and the intrinsic properties of phosphoglucoisomerase, was first examined. According to a theoretical model with enzyme-to-enzyme channelling, the generation of 3HOH from D-[2-3H]glucose should be higher in islets exposed to beta-D-glucose rather than alpha-D-glucose, whilst the opposite situation should prevail in the case of D-[5-3H]glucose conversion to 3HOH. Experimental data collected in rat islets incubated for 60 min at 4 degrees C in the presence of either alpha- or beta-D-glucose mixed with tracer amounts of either alpha- or beta-D-[2- 3H]glucose and alpha- or beta-D-[5-3H]glucose indicate that the beta/alpha ratio for D-[2-3H]glucose conversion to 3HOH is indeed higher than the beta/alpha ratio for D-[5-3H]glucose conversion to 3HOH. These findings are consistent with the postulated enzyme-to-enzyme tunnelling of glycolytic intermediates between hexokinase isoenzyme(s), phosphoglucoisomerase and, possibly, phosphofructokinase.     

Hexose metabolism in pancreatic islets: enzyme-to-enzyme tunnelling of hexose 6-phosphates.

The fate of unlabelled D-glucose and D-[2-3H]glucose in pancreatic islets was simulated taking into account experimental values for glycolytic flux, intracellular concentration of D-glucose 6-phosphate and phosphoglucoisomerase activity. The model, which also takes into account the isotopic discrimination in velocity and intramolecular transfer of tritium between D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase, revealed that the predicted generation of 3HOH from D-[2-3H]glucose was much higher than the true experimental value. Such a discrepancy is reinforced by the consideration that the generation of 3HOH from D-[2-3H]glucose in islet cells is not solely attributable to the phosphoglucoisomerase-catalyzed detritiation of hexose 6-phosphates metabolized in the glycolytic pathway. In order to reconcile experimental and theoretical values for 3HOH production, it was found necessary to postulate enzyme-to-enzyme tunnelling of hexose 6-phosphates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. It is proposed that such a tunnelling may favour the anomeric specificity of D-glucose metabolism in islet cells, by restricting the anomerization of hexose 6-phosphates.     

Environmental modulation of the anomeric specificity of glucose metabolism in normal and tumoral cells

In rat pancreatic islets and erythrocytes, alpha-D-glucose (2.8-5.6 mM) is better metabolized than beta-D-glucose, as judged from the conversion of D-[5-3H]glucose to 3H2O, augmentation in lactic acid production (or output) or oxidation of D-[U-14C]glucose. In tumoral cells, however, whether of the insulin-producing or lymphocytic leukemia type, the anomeric preference for alpha-D-glucose utilization is no longer present when the cells are incubated at comparable glucose concentrations (2.8-4.0 mM). Nevertheless, the tumoral insulin-producing cells are able to display preference for either alpha-D-glucose (at very low glucose concentrations in the 0.14-0.82 mM range) or beta-D-glucose (in the presence of 16.7 mM glucose). These findings indicate that the anomeric specificity of glucose metabolism may differ in distinct cell types, and can be modulated by the ambient glucose concentration. ambient glucose concentration.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates. A model for D-[2-3H]glucose metabolism in human erythrocytes

When D-[2-3H]glucose 6-phosphate mixed with the unlabeled ester is converted to D-[1-3H]fructose 6-phosphate and 3HOH in the phosphoglucoisomerase reaction and then to D-[1-3H]fructose 1,6-bisphosphate in the phosphofructokinase reaction, the specific radioactivity of the latter metabolite and the production of 3HOH relative to the total generation of tritiated end products are both inversely related to the concentration of phosphofructokinase. In human erythrocytes, the modeling of D-[2-3H]glucose metabolism, based on the activity of phosphoglucoisomerase in cell homogenates and on the steady-state content of D-glucose 6-phosphate and D-fructose 6-phosphate in intact cells, indicates that the back-and-forth interconversion of these esters is about five-times higher than the net glycolytic flux. Yet, the production of 3HOH from D-[2-3H]glucose is about 20% lower than the net glycolytic flux, as judged from the production of 3HOH from D-[5-3H]glucose. Thus, an incomplete detriation of D-[2-3H]glucose is not incompatible with an extensive interconversion of hexose 6-phosphates in the reaction catalyzed by phosphoglucoisomerase.     

Hexose metabolism in pancreatic islets. Insignificance of D-glucose futile cycling in rat islets.

When rat pancreatic islets were incubated in the presence of unlabelled D-glucose (16.7 mM) and 3HOH, the production of 3H-labelled material susceptible to be phosphorylated by yeast hexokinase and then detritiated by yeast phosphoglucoisomerase did not exceed 2.66 +/- 0.21 pmol/islet per 180 min, i.e. about 1% of the rate of exogenous D-[5-3H]glucose utilization. Such a material accounted for 43 +/- 4% of the total radioactivity, associated with tritiated hexose(s). It is proposed, therefore, that the futile cycling of D-glucose in the reactions catalyzed in the islet cells by the hexokinase isoenzymes and glucose-6-phosphatase represents a negligible fraction of the total rate of D-glucose phosphorylation.     

Anomeric dissociation between glucokinase activity and glycolysis in pancreatic islets

In pancreatic islet homogenates incubated in the presence of a high glucose concentration (40 mM), the beta-anomer of D-glucose is phosphorylated at a higher rate than the alpha-anomer, whether in the absence or presence of exogenous glucose 6-phosphate. However, in intact islets also exposed to 40 mM D-glucose, the production of 3H2O from D-[5-3H] glucose, the oxidation of D-[U-14C] glucose and the glucose-induced increment in either lactate production or 45Ca net uptake, as well as the release of insulin from isolated perfused pancreases, are not higher with beta- than alpha-D-glucose. It is concluded that the rate of glucose utilization by islet cells is not regulated solely by the activity of hexokinase and/or glucokinase.     

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 D-glucose metabolism in rat adipocytes

The anomeric specificity of D-glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8 degrees C to pure alpha- or beta-D-glucose or to equilibrated D-glucose. The rate of D-[5-3H]glucose utilization was higher with alpha- than beta-D-glucose. However, as judged from the oxidation of D-[1-14C]glucose and D-[6-14C]glucose anomers, the fraction of D-glucose catabolism occurring via the pentose cycle was higher with beta- than alpha-D-glucose. In the presence of equilibrated D-glucose, the utilization of alpha-D-[5-3H]glucose and the oxidation of both alpha-D-[1-14C]glucose and alpha-D-[6-14C]glucose were higher, relative to the anomer concentration, than the corresponding values for beta-D-glucose. It is concluded that the anomeric specificity of D-glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D-glucose.     

Anomeric specificity of glycolysis in a non glucokinase-containing cell

In rat erythrocyte homogenates, the phosphorylation of D-glucose measured at 30 degrees C over a wide range of glucose concentrations (50 microM to 20 mM) yielded in a double reciprocal plot a single straight line with a Km close to 0.06 mM and a maximal velocity close to 47 nmol/60 min per mg hemoglobin. At 8 degrees C, the rate of glucose phosphorylation was 60% higher in the presence of beta-D-glucose than alpha-D-glucose. Yet, in intact erythrocytes incubated at 8 degrees C in the presence of beta-D-glucose (4 or 7 mM), the glucose-induced increment in lactic acid output represented no more than 39 to 74% of that found in the presence of alpha-D-glucose. Thus, a greater rate of glycolysis in the presence of alpha-D-glucose was observed in a cell devoid of glucokinase and containing a hexokinase with preference for beta-D-glucose. These findings indicate that the anomeric specificity of glycolysis in intact cells cannot be predicted and does not necessarily depend on the anomeric preference of glucose-phosphorylating enzyme(s).     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.     

Comparison between D-[3-3H]- and D-[5-3H]glucose and fructose utilization in pancreatic islets from control and hereditarily diabetic rats

The metabolism of D-glucose and/or D-fructose was investigated in pancreatic islets from control rats and hereditarily diabetic GK rats. In the case of both D-glucose and D-fructose metabolism, a preferential alteration of oxidative events was observed in islets from GK rats. The generation of 3HOH from D-[5-3H]glucose (or D-[5-3H]fructose) exceeded that from D-[3-3H]glucose (or D-[3-3H]fructose) in both control and GK rats. This difference, which is possibly attributable to a partial escape from glycolysis of tritiated dihydroxyacetone phosphate, was accentuated whenever the rate of glycolysis was decreased, e.g., in the absence of extracellular Ca(2+) or presence of exogenous D-glyceraldehyde. D-Mannoheptulose, which inhibited D-glucose metabolism, exerted only limited effects upon D-fructose metabolism. In the presence of both hexoses, the paired ratio between D-[U-14C]fructose oxidation and D-[3-3H]fructose or D-[5-3H]fructose utilization was considerably increased, this being probably attributable, in part at least, to a preferential stimulation by the aldohexose of mitochondrial oxidative events. Moreover, this coincided with the fact that D-mannoheptulose now severely inhibited the catabolism of D-[5-3H]fructose and D-[U-14C]fructose. The latter situation is consistent with both the knowledge that D-glucose augments D-fructose phosphorylation by glucokinase and the findings that D-mannoheptulose, which fails to affect D-fructose phosphorylation by fructokinase, inhibits the phosphorylation of D-fructose by glucokinase.     

Generation of 3HOH from D-[6-3H]glucose by erythrocytes: role of pyruvate alanine interconversion

Human and rat erythrocytes were found to generate 3HOH from D-[6(N)-3H]glucose. The rate of 3HOH production represented 7-10% of the glycolytic flux. The generation of 3HOH appeared attributable, in part at least, to the detritiation of [3-3H]pyruvate during the interconversion of the 2-keto acid and L-alanine in the reaction catalyzed by glutamate-pyruvate transaminase. Indeed, purified pig heart glutamate-pyruvate transaminase, as well as homogenates prepared from rat erythrocytes or pancreatic islets, catalyzed the generation of 3HOH from L-[3-3H]alanine. When the production of tritiated pyruvate from L-[3-3H]alanine was coupled to the conversion of the 2-keto acid to L-lactate, the production of 3HOH accounted for one-third of the reaction velocity, the latter failing to display isotopic discrimination. In these experiments, the production of 3HOH was abolished by amino-oxyacetate. Likewise, in intact rat erythrocytes, aminooxyacetate inhibited the generation of 3HOH and tritiated L-alanine from D-[6-3H]glucose (or D-[1-3H]glucose), as well as the generation of 3HOH from L-[3-3H]alanine. In pancreatic islets, however, aminooxyacetate failed to affect significantly the generation of 3HOH from D-[6-3H]glucose. These findings indicate that the generation of 3HOH from D-[6-3H]glucose is mainly attributable to an intermolecular tritium transfer in transaminase reaction, at least in cells devoid of mitochondria.     

Anomeric specificity of D-glucose phosphorylation and oxidation in human erythrocytes

1. In human erythrocytes, alpha-D-[U-14C]glucose is more efficiently oxidized than beta-D-[U-14C]glucose at a low concentration of the hexose (0.1 mM), but not so at higher glucose concentrations. 2. This unexpected situation may be attributable in part to the lower Km of hexokinase for alpha- than beta-D-glucose, this difference in affinity compensating for the higher maximal velocity found with the beta- rather than alpha-anomer. 3. A contributive role for aldose reductase in the anomeric control of D-glucose 6-phosphate circulation in the pentose phosphate pathway should not be ruled out, since aldose reductase inhibitors decrease the production of 14CO2 by erythrocytes exposed to D-[U-14C]glucose. 4. Nevertheless, the essential role of hexokinase in such an anomeric control is supported by the finding that, in the presence of menadione, which augments considerably D-[U-14C]glucose oxidation but fails to affect D-[5-3H]glucose utilization, the anomeric alpha/beta ratio in 14CO2 production from D-[U-14C]glucose follows, at increasing concentrations of the hexose, the same pattern as that found for its phosphorylation.     

Anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms

The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.     

Preferential stimulation by glucose of its oxidation relative to glycolysis in purified insulin-producing cells.

A rise in extracellular D-glucose concentration increases to a greater relative extent the conversion of both D-[5-3H]glucose to 3HOH and D-[6-14C]glucose to 14CO2 in rat purified insulin-producing cells than previously observed in pancreatic islets. In the pure B-cells, the ratio between D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization increases, in a sigmoidal manner, as a function of the hexose concentration. The preferential stimulation by D-glucose of mitochondrial oxidative events is proposed to represent an unusual but essential feature of the metabolic and, hence, functional response of these fuel-sensor cells.     

Dual anomeric specificity and dual anomerase activity of phosphoglucoisomerase quantified by two-dimensional phase-sensitive 13C EXSY NMR.

The reversible conversion between D-glucose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphoglucoisomerase was studied by phase sensitive two-dimensional 13C-[1H] EXSY NMR spectroscopy at 150.869 and 125.759 MHz, using 13C-enriched substrates in the C2 position of the D-hexose 6-phosphates. The shape of the build-up curves of the cross-peaks associated with the 13C2 resonances of the alpha- and beta-anomers of both D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate reveals that phosphoglucoisomerase selectively catalyzes the reversible conversion between alpha-D-[2-13C]glucose 6-phosphate and beta-D-[2-13C]fructose 6-phosphate. Quantitative analysis of the build-up curves by three different methods allowed us to conclude that phosphoglucoisomerase not only selectively channels the latter isomerization but also considerably accelerates the anomerization of both D-hexose 6-phosphates. The rate constants of anomerization were indeed much higher in the presence than in the absence of enzyme. The major finding in the present study consists in the anomeric specificity of phosphoglucoisomerase toward the beta-anomer of D-fructose 6-phosphate both as a substrate and a product, contrary to previous proposals. This finding supports recent evidence suggesting the direct channelling of beta-D-fructose 6-phosphate from phosphoglucoisomerase to phosphofructokinase.     

Anomeric specificity of D-glucose production by rat hepatocytes

The anomeric specificity of D-glucose metabolism in intact hepatocytes remains a matter of debate. This issue was further investigated in the present study, which is based on the quantification of the alpha- and beta-anomers of the 13C-enriched isotopomers of D-glucose generated by rat liver cells exposed to either D-[1-13C] fructose or D-[2-13C] fructose in the presence of D2O. The D-[1-13C]glucose/D-[6-13C]glucose paired ratios found in the cells exposed to D-[1-13C] fructose and the D-[2-13C]glucose/D-[5-13C]glucose paired ratios found in the cells exposed to D-[2-13C] fructose yielded a paired beta/alpha ratio averaging (mean +/- S.E.M.) 79.3 +/- 6.1%. In the case of the isotopomers of D-glucose formed by gluconeogenesis, the D-[2-13C]glucose/D-[5-13C]glucose and D-[3-13C]glucose/D-[4-13C]glucose paired ratios found in cells exposed to D-[1-13C] fructose, as well as the D-[1-13C]glucose/D-[6-13C]glucose and D-[3-13C]glucose/D-[4-13C]glucose paired ratios found in cells exposed to D-[2-13C]fructose, yielded an alpha/beta paired ratio averaging 75.0 +/- 5.8%. Last, in the cells exposed to D-[2-13C]fructose, the beta/alpha ratio for the C2-deuterated isotopomers of D-[2-13C]glucose represented 78.9 +/- 3.7% of that for the C5-deuterated isotopomers of D-[5-13C]glucose. The three values representative of the anomeric specificity of D-glucose production by liver cells were not significantly different from one another, with an overall mean value of 76.9 +/- 3.6%. These findings unambiguously document that the anomeric specificity of phosphoglucoisomerase is operative in intact hepatocytes, resulting in a preferential output of the alpha-anomer of 13C-enriched D-glucose under the present experimental conditions.     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.