Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the... more Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling ...
International journal of pancreatology : official journal of the International Association of Pancreatology, 1998
The polyacetate esters of selected nonnutrient monosaccharides represent potential tools for eith... more The polyacetate esters of selected nonnutrient monosaccharides represent potential tools for either stimulation of insulin release in noninsulin-dependent diabetes or inhibition of insulin secretion in hyperinsulinemic syndromes. The polyacetate esters of several monosaccharides were recently shown to display greater nutritional value or biological efficiency than the corresponding unesterified carbohydrates. The effects of seven polyacetate esters of monosaccharides, all tested at a 1.7-mM concentration, on both 45Ca efflux and insulin release were investigated in prelabeled rat pancreatic islets perifused in the presence of 10.0 mM succinic acid dimethyl ester. Both alpha-D-glucose penta-acetate and, to a lesser extent, beta-L-glucose penta-acetate stimulated insulin release. Inversely, alpha-D-galactose penta-acetate, but not beta-D-galactose penta-acetate inhibited insulin secretion evoked by succinic acid dimethyl ester. Esters of carbohydrates which are inhibitors of D-glucose...
1. Selected esters of succinic acid are currently under investigation as potential insulinotropic... more 1. Selected esters of succinic acid are currently under investigation as potential insulinotropic tools in the treatment of non-insulin-dependent diabetes. At variance with the methyl esters of succinic acid used in most of the work so far conducted from this perspective, the monoethyl ester of succinic acid (EMS) offers the advantage of avoiding the undesirable generation of methanol by intracellular hydrolysis. In the present study, the metabolism and functional effects of EMS were investigated, therefore, in rat pancreatic islets. 2. At a 10 mM concentration, EMS enhanced insulin release from islets stimulated by 7-17 mM D-glucose but failed to do so at lower concentrations of the hexose. EMS was efficiently metabolized, as judged from the generation of 14CO2 by islets exposed to the monoethyl ester of either [1,4-14C] or [2, 3-14C]succinic acid. D-Glucose (6 mM) failed to affect the metabolism of EMS (10 mM), which itself failed to affect the metabolism of D-[5-3H]glucose or D-[U-14C]glucose. EMS also stimulated biosynthetic activity in the islets. It inhibited 86Rb and 45Ca outflow from prelabeled islets perfused in the absence of D-glucose but enhanced the efflux of the two cationic tracers in the presence of the hexose (7 mM). 3. It is concluded that the insulinotropic action of EMS is attributable, to a large extent, to its capacity to act as a nutrient in islet cells.
Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the... more Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.
The dimethyl esters of succinic acid (SAD) and glutamic acid (GME) were found to be efficiently m... more The dimethyl esters of succinic acid (SAD) and glutamic acid (GME) were found to be efficiently metabolized in colon carcinoma cells of the Caco-2 line. The rate of [1,4-14C]SAD and [2,3-14C]SAD conversion to radioactive acidic metabolites, CO2, amino acids, pyruvic acid, and lactic acid suggested that the catabolism of the ester-derived succinic acid occurred mainly through the sequence of reactions catalyzed by succinate dehydrogenase, fumarase, and the malic enzyme. This coincided with a marked sparing action of SAD on the utilization of D-[2-(3)H]glucose and D-[5-(3)H]glucose and generation of 14C-labeled acid metabolites, CO2, and lactic acid from D-[U-14C]glucose by the enterocytes. Likewise, the conversion of [U-14C]GME to 14C-labeled amino acids, its oxidation compared with that of [1-(14)C]GME, and the production of NH4+ in the absence or presence of GME indicated efficient catabolism of the latter ester. Like SAD, GME decreased the utilization of D-[5-(3)H]glucose and generation of 14C-labeled acidic metabolites, pyruvate, and CO2 from D-[6-(14)C]glucose, while increasing the generation of 14C-labeled amino acids from the labeled hexose. The oxidation of D-[6-(14)C]glucose was even more severely inhibited by GME. In normal rat intestinal cells, SAM, SAD, and GME also exerted a marked sparing action on D-[U-14C]glucose oxidation. The present findings suggest, therefore, that these esters could possibly be used to sustain ATP generation in intestinal cells.
... The incorporation ofl-[4-3 H]phenylalanine into TCA-precipitable material observed in the pre... more ... The incorporation ofl-[4-3 H]phenylalanine into TCA-precipitable material observed in the presence of both 4.2 mMd-glucose and 1.7 mM α-d-glucose pentaacetate was comparable to that otherwise found in the sole presence of 7.0 mMd-glucose; it remained significantly lower ...
D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-produci... more D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-producing cells in the pancreatic gland. In the present study, D-[3H]-mannoheptulose uptake by rat pancreatic islets or dispersed islet cells was found to represent a time-related and temperature-sensitive process inhibited by cytochalasin B. This mould metabolite also inhibited the efflux of D-[3H]-mannoheptulose from prelabelled islets. After 60 min incubation at 37 degrees C, the apparent intracellular distribution space of the tritiated heptose was close to or somewhat higher than that of D-[5-3H]glucose and close to 50% of the intracellular 3HOH space. It was further enhanced by D-glucose and a high concentration of 10 mM of D-mannoheptulose. The uptake of D-[3H]mannoheptulose was much lower however than that of D-[3H]mannoheptulose hexaacetate. As judged from the fate of D-mannoheptulose hexa[2-14C]acetate, the latter ester was efficiently hydrolyzed in the islet cells. The internalization of D-[3H]mannoheptulose (or its ester) coincided with the generation of tritiated acidic metabolites, reflecting phosphorylation of the heptose. The situation found in normal islet cells sharply differed from that found in tumoral islet cells of either the RINm5F or INS-1 line, in which the apparent distribution space of D-[3H]mannoheptulose represented only about 3 and 9%, respectively, of the intracellular 3HOH space. These results indicate that the entry of D-mannoheptulose into islet cells represents a carrier-mediated process, possibly mediated at the intervention of GLUT2 and, hence, provide further support to the possible use of a suitable D-mannoheptulose analog as a tool for the preferential labelling of insulin-producing cells in the pancreatic gland.
The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously ... more The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously documented. However, no information was so far available on the activity of this enzyme in islets relative to that in other tissues and on the respective contribution of insulin-producing B cells and non-B islet cells. The present study provides such an information. The activity of fructokinase, as assessed by the phosphorylation of 1.0 mM D-fructose, was compared to that of hexokinase isoenzyme(s), as measured in the presence of 1.0 mM D-glucose, and further characterized by its heat-resistance, K+ dependency and resistance to the inhibitory action of D-mannoheptulose. As judged from the results obtained in heated homogenates, the activity of fructokinase, expressed relative to protein content (nmol/min per mg protein) was highest in liver (21.5 +/- 2.5; n = 11) and lowest in parotid gland (0.16 +/- 0.09; n = 3), with in-between values in ileum (2.45 +/- 0.53; n = 3), pancreas (0.82 +/-...
D-glucose was previously reported to cause a concentration-related decrease in the 36Cl- content ... more D-glucose was previously reported to cause a concentration-related decrease in the 36Cl- content of prelabeled islets prepared from ob/ob mice, a current animal model of inherited obesity. From these findings, it was inferred that the hexose stimulates Cl- efflux from islet cells and that such an increase in Cl- permeability may partly mediate glucose-induced depolarization of insulin-producing cells. The aim of the present study was to investigate the possible extension of these findings to islets prepared from normal rats by measuring the changes evoked by increasing concentrations of D-glucose in 36Cl- outflow itself from prelabeled isolated islets. After 60 min preincubation at 37 degrees C in the presence of 3 mM D-glucose and 36Cl- (75 microCi/mL), the islets were incubated for 8-10 min at 37 degrees C in the presence of increasing concentrations of the hexose (3-20 mM). The changes in 36Cl- outflow during incubation indicated that D-glucose, in excess of a threshold concentra...
The international journal of biochemistry & cell biology, 2004
The metabolism of D-glucose displays anomeric specificity in rat pancreatic islets. The aim of th... more 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]...
In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) ... more In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-14C]glucose than D-[6-14C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired 14C/3H ratio found in islets exposed to both D-[1-14C]glucose or D-[6-14C]glucose and D-[3-3H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-14C]glucose. The labelling of islet lipids by D-[6-14C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphat...
Cytochalasin B is known to enhance insulin release evoked by nutrient and non-nutrient secretagog... more Cytochalasin B is known to enhance insulin release evoked by nutrient and non-nutrient secretagogues, including D-glucose, despite inhibiting D-glucose uptake and metabolism in pancreatic islets. In the present study, cytochalasin D, which failed to affect D-glucose uptake and metabolism by isolated islets, also augmented glucose-stimulated insulin release, but unexpectedly to a lesser extent than cytochalasin B. Such was not the case, however, in islets stimulated by non-glucidic nutrients such as 2-ketoisocaproate or the association of L-leucine and L-glutamine. This situation coincided with the fact that cytochalasin B inhibited more severely D-glucose metabolism in non-B, as distinct from B, islet cells and, in the former case, caused a relatively greater inhibition of hexose catabolism at 2.8 mM than at 16.7 mM D-glucose. Nevertheless, even in the presence of forskolin, cytochalasin B was more efficient than cytochalasin D in augmenting glucose-stimulated insulin secretion. Thu...
D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of... more D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of GLUT2. In the present study, the heptose (10 mM) decreased the steady state content of dispersed rat pancreatic islet cells in D-[U-(14)C]glucose, and inhibited to a greater relative extent the utilization of D-[5-(3)H]glucose, the oxidation of D-[U-(14)C]-glucose and its conversion to radioactive amino acid when the dispersed islet cells were incubated at 16.7 mM rather than 2.8 mM D-glucose. A comparable situation was found in purified islet B-cells, whereas D-mannoheptulose only exerted minor to negligible effects upon the metabolism of D-glucose in non-B islet cells. This coincided with a much higher uptake of D-[(3)H]mannoheptulose by B, as distinct from non-B, islet cells. These findings indicate that the unexpectedly greater relative inhibitory action of D-mannoheptulose upon D-glucose metabolism by isolated islets (or dispersed islet cells) observed at high rather than low hexos...
D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells ... more D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells in the pancreatic gland in the perspective of the non-invasive imaging of the endocrine pancreas. In such a perspective, we have now synthesized 1-deoxy-1-[125I]iodo-D-mannoheptulose ([125I]MH) and examined its uptake by different rat cell types. No phosphorylation of [125I]MH by bovine heart hexokinase could be detected. The apparent distribution space of [125I]MH largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, in erythrocytes, parotid cells, hepatocytes, pancreatic pieces and isolated pancreatic islets. Relative to the mean intracellular distribution space of 3HOH, that of [125I]MH was not significantly different in pancreatic pieces from either normal rats or streptozotocin-induced diabetic animals (STZ rats). In pancreatic islets, the uptake of [125I]MH was decreased at low temperature, but failed to be significantly affected by cytochalasin B. Sixty m...
Diadenosine polyphosphates, such as diadenosine triphosphate (A2P3) and diadenosine tetraphosphat... more Diadenosine polyphosphates, such as diadenosine triphosphate (A2P3) and diadenosine tetraphosphate (A2P4), were recently proposed to participate in the stimulus-secretion coupling for nutrient-stimulated insulin release. Since NaF, an inhibitor of inorganic pyrophosphatase, was reported to lower A2P3 and A2P4 content in glucose-stimulated pancreatic islets, its effects upon metabolic, cationic, biosynthetic and secretory variables in rat pancreatic islets were investigated in the present study. Up to a concentration close to 0.1 mM, NaF failed to affect most of these variables, except for a decrease in 45Ca net uptake. Much higher concentrations of NaF (e.g. 5.0 mM) were required to cause inhibition of the metabolic, ionic, biosynthetic and secretory responses of the islets to nutrient secretagogues. Yet, even at this high concentration, NaF failed to lower the islet content in tritiated A2P3 and A2P4 in islets prelabelled with [2,8-3H]adenosine and failed to prevent the glucose-ind...
The metabolism of alpha-D-glucose pentaacetate and its positive insulinotropic action in isolated... more The metabolism of alpha-D-glucose pentaacetate and its positive insulinotropic action in isolated rat pancreatic islets are both unexpectedly resistant to D-mannoheptulose, as judged from experiments conducted over 90-120 min incubation. In the present study, the possible effects of the heptose upon the immediate cationic and secretory response to the ester were investigated in perifused islets prelabeled with either 86Rb or 45Ca. At a 10 mM concentration, sufficient to abolish the inhibitory action of unesterified D-glucose upon 86Rb outflow, D-mannoheptulose failed to suppress the decrease in 86Rb outflow and increase in 45Ca efflux caused by alpha-D-glucose pentaacetate at normal extracellular Ca2+ concentration and also failed to prevent the decrease in both 45Ca and insulin release provoked by the ester in the absence of extracellular Ca2+. The sole obvious effect of the heptose was to change the early peak-shaped positive secretory response to alpha-D-glucose pentaacetate to a...
The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose... more The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5...
Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the... more Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling ...
International journal of pancreatology : official journal of the International Association of Pancreatology, 1998
The polyacetate esters of selected nonnutrient monosaccharides represent potential tools for eith... more The polyacetate esters of selected nonnutrient monosaccharides represent potential tools for either stimulation of insulin release in noninsulin-dependent diabetes or inhibition of insulin secretion in hyperinsulinemic syndromes. The polyacetate esters of several monosaccharides were recently shown to display greater nutritional value or biological efficiency than the corresponding unesterified carbohydrates. The effects of seven polyacetate esters of monosaccharides, all tested at a 1.7-mM concentration, on both 45Ca efflux and insulin release were investigated in prelabeled rat pancreatic islets perifused in the presence of 10.0 mM succinic acid dimethyl ester. Both alpha-D-glucose penta-acetate and, to a lesser extent, beta-L-glucose penta-acetate stimulated insulin release. Inversely, alpha-D-galactose penta-acetate, but not beta-D-galactose penta-acetate inhibited insulin secretion evoked by succinic acid dimethyl ester. Esters of carbohydrates which are inhibitors of D-glucose...
1. Selected esters of succinic acid are currently under investigation as potential insulinotropic... more 1. Selected esters of succinic acid are currently under investigation as potential insulinotropic tools in the treatment of non-insulin-dependent diabetes. At variance with the methyl esters of succinic acid used in most of the work so far conducted from this perspective, the monoethyl ester of succinic acid (EMS) offers the advantage of avoiding the undesirable generation of methanol by intracellular hydrolysis. In the present study, the metabolism and functional effects of EMS were investigated, therefore, in rat pancreatic islets. 2. At a 10 mM concentration, EMS enhanced insulin release from islets stimulated by 7-17 mM D-glucose but failed to do so at lower concentrations of the hexose. EMS was efficiently metabolized, as judged from the generation of 14CO2 by islets exposed to the monoethyl ester of either [1,4-14C] or [2, 3-14C]succinic acid. D-Glucose (6 mM) failed to affect the metabolism of EMS (10 mM), which itself failed to affect the metabolism of D-[5-3H]glucose or D-[U-14C]glucose. EMS also stimulated biosynthetic activity in the islets. It inhibited 86Rb and 45Ca outflow from prelabeled islets perfused in the absence of D-glucose but enhanced the efflux of the two cationic tracers in the presence of the hexose (7 mM). 3. It is concluded that the insulinotropic action of EMS is attributable, to a large extent, to its capacity to act as a nutrient in islet cells.
Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the... more Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.
The dimethyl esters of succinic acid (SAD) and glutamic acid (GME) were found to be efficiently m... more The dimethyl esters of succinic acid (SAD) and glutamic acid (GME) were found to be efficiently metabolized in colon carcinoma cells of the Caco-2 line. The rate of [1,4-14C]SAD and [2,3-14C]SAD conversion to radioactive acidic metabolites, CO2, amino acids, pyruvic acid, and lactic acid suggested that the catabolism of the ester-derived succinic acid occurred mainly through the sequence of reactions catalyzed by succinate dehydrogenase, fumarase, and the malic enzyme. This coincided with a marked sparing action of SAD on the utilization of D-[2-(3)H]glucose and D-[5-(3)H]glucose and generation of 14C-labeled acid metabolites, CO2, and lactic acid from D-[U-14C]glucose by the enterocytes. Likewise, the conversion of [U-14C]GME to 14C-labeled amino acids, its oxidation compared with that of [1-(14)C]GME, and the production of NH4+ in the absence or presence of GME indicated efficient catabolism of the latter ester. Like SAD, GME decreased the utilization of D-[5-(3)H]glucose and generation of 14C-labeled acidic metabolites, pyruvate, and CO2 from D-[6-(14)C]glucose, while increasing the generation of 14C-labeled amino acids from the labeled hexose. The oxidation of D-[6-(14)C]glucose was even more severely inhibited by GME. In normal rat intestinal cells, SAM, SAD, and GME also exerted a marked sparing action on D-[U-14C]glucose oxidation. The present findings suggest, therefore, that these esters could possibly be used to sustain ATP generation in intestinal cells.
... The incorporation ofl-[4-3 H]phenylalanine into TCA-precipitable material observed in the pre... more ... The incorporation ofl-[4-3 H]phenylalanine into TCA-precipitable material observed in the presence of both 4.2 mMd-glucose and 1.7 mM α-d-glucose pentaacetate was comparable to that otherwise found in the sole presence of 7.0 mMd-glucose; it remained significantly lower ...
D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-produci... more D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-producing cells in the pancreatic gland. In the present study, D-[3H]-mannoheptulose uptake by rat pancreatic islets or dispersed islet cells was found to represent a time-related and temperature-sensitive process inhibited by cytochalasin B. This mould metabolite also inhibited the efflux of D-[3H]-mannoheptulose from prelabelled islets. After 60 min incubation at 37 degrees C, the apparent intracellular distribution space of the tritiated heptose was close to or somewhat higher than that of D-[5-3H]glucose and close to 50% of the intracellular 3HOH space. It was further enhanced by D-glucose and a high concentration of 10 mM of D-mannoheptulose. The uptake of D-[3H]mannoheptulose was much lower however than that of D-[3H]mannoheptulose hexaacetate. As judged from the fate of D-mannoheptulose hexa[2-14C]acetate, the latter ester was efficiently hydrolyzed in the islet cells. The internalization of D-[3H]mannoheptulose (or its ester) coincided with the generation of tritiated acidic metabolites, reflecting phosphorylation of the heptose. The situation found in normal islet cells sharply differed from that found in tumoral islet cells of either the RINm5F or INS-1 line, in which the apparent distribution space of D-[3H]mannoheptulose represented only about 3 and 9%, respectively, of the intracellular 3HOH space. These results indicate that the entry of D-mannoheptulose into islet cells represents a carrier-mediated process, possibly mediated at the intervention of GLUT2 and, hence, provide further support to the possible use of a suitable D-mannoheptulose analog as a tool for the preferential labelling of insulin-producing cells in the pancreatic gland.
The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously ... more The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously documented. However, no information was so far available on the activity of this enzyme in islets relative to that in other tissues and on the respective contribution of insulin-producing B cells and non-B islet cells. The present study provides such an information. The activity of fructokinase, as assessed by the phosphorylation of 1.0 mM D-fructose, was compared to that of hexokinase isoenzyme(s), as measured in the presence of 1.0 mM D-glucose, and further characterized by its heat-resistance, K+ dependency and resistance to the inhibitory action of D-mannoheptulose. As judged from the results obtained in heated homogenates, the activity of fructokinase, expressed relative to protein content (nmol/min per mg protein) was highest in liver (21.5 +/- 2.5; n = 11) and lowest in parotid gland (0.16 +/- 0.09; n = 3), with in-between values in ileum (2.45 +/- 0.53; n = 3), pancreas (0.82 +/-...
D-glucose was previously reported to cause a concentration-related decrease in the 36Cl- content ... more D-glucose was previously reported to cause a concentration-related decrease in the 36Cl- content of prelabeled islets prepared from ob/ob mice, a current animal model of inherited obesity. From these findings, it was inferred that the hexose stimulates Cl- efflux from islet cells and that such an increase in Cl- permeability may partly mediate glucose-induced depolarization of insulin-producing cells. The aim of the present study was to investigate the possible extension of these findings to islets prepared from normal rats by measuring the changes evoked by increasing concentrations of D-glucose in 36Cl- outflow itself from prelabeled isolated islets. After 60 min preincubation at 37 degrees C in the presence of 3 mM D-glucose and 36Cl- (75 microCi/mL), the islets were incubated for 8-10 min at 37 degrees C in the presence of increasing concentrations of the hexose (3-20 mM). The changes in 36Cl- outflow during incubation indicated that D-glucose, in excess of a threshold concentra...
The international journal of biochemistry & cell biology, 2004
The metabolism of D-glucose displays anomeric specificity in rat pancreatic islets. The aim of th... more 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]...
In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) ... more In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-14C]glucose than D-[6-14C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired 14C/3H ratio found in islets exposed to both D-[1-14C]glucose or D-[6-14C]glucose and D-[3-3H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-14C]glucose. The labelling of islet lipids by D-[6-14C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphat...
Cytochalasin B is known to enhance insulin release evoked by nutrient and non-nutrient secretagog... more Cytochalasin B is known to enhance insulin release evoked by nutrient and non-nutrient secretagogues, including D-glucose, despite inhibiting D-glucose uptake and metabolism in pancreatic islets. In the present study, cytochalasin D, which failed to affect D-glucose uptake and metabolism by isolated islets, also augmented glucose-stimulated insulin release, but unexpectedly to a lesser extent than cytochalasin B. Such was not the case, however, in islets stimulated by non-glucidic nutrients such as 2-ketoisocaproate or the association of L-leucine and L-glutamine. This situation coincided with the fact that cytochalasin B inhibited more severely D-glucose metabolism in non-B, as distinct from B, islet cells and, in the former case, caused a relatively greater inhibition of hexose catabolism at 2.8 mM than at 16.7 mM D-glucose. Nevertheless, even in the presence of forskolin, cytochalasin B was more efficient than cytochalasin D in augmenting glucose-stimulated insulin secretion. Thu...
D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of... more D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of GLUT2. In the present study, the heptose (10 mM) decreased the steady state content of dispersed rat pancreatic islet cells in D-[U-(14)C]glucose, and inhibited to a greater relative extent the utilization of D-[5-(3)H]glucose, the oxidation of D-[U-(14)C]-glucose and its conversion to radioactive amino acid when the dispersed islet cells were incubated at 16.7 mM rather than 2.8 mM D-glucose. A comparable situation was found in purified islet B-cells, whereas D-mannoheptulose only exerted minor to negligible effects upon the metabolism of D-glucose in non-B islet cells. This coincided with a much higher uptake of D-[(3)H]mannoheptulose by B, as distinct from non-B, islet cells. These findings indicate that the unexpectedly greater relative inhibitory action of D-mannoheptulose upon D-glucose metabolism by isolated islets (or dispersed islet cells) observed at high rather than low hexos...
D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells ... more D-mannoheptulose was recently proposed as a tool to label preferentially insulin-producing cells in the pancreatic gland in the perspective of the non-invasive imaging of the endocrine pancreas. In such a perspective, we have now synthesized 1-deoxy-1-[125I]iodo-D-mannoheptulose ([125I]MH) and examined its uptake by different rat cell types. No phosphorylation of [125I]MH by bovine heart hexokinase could be detected. The apparent distribution space of [125I]MH largely exceeded that of [U-14C]sucrose, considered as an extracellular marker, in erythrocytes, parotid cells, hepatocytes, pancreatic pieces and isolated pancreatic islets. Relative to the mean intracellular distribution space of 3HOH, that of [125I]MH was not significantly different in pancreatic pieces from either normal rats or streptozotocin-induced diabetic animals (STZ rats). In pancreatic islets, the uptake of [125I]MH was decreased at low temperature, but failed to be significantly affected by cytochalasin B. Sixty m...
Diadenosine polyphosphates, such as diadenosine triphosphate (A2P3) and diadenosine tetraphosphat... more Diadenosine polyphosphates, such as diadenosine triphosphate (A2P3) and diadenosine tetraphosphate (A2P4), were recently proposed to participate in the stimulus-secretion coupling for nutrient-stimulated insulin release. Since NaF, an inhibitor of inorganic pyrophosphatase, was reported to lower A2P3 and A2P4 content in glucose-stimulated pancreatic islets, its effects upon metabolic, cationic, biosynthetic and secretory variables in rat pancreatic islets were investigated in the present study. Up to a concentration close to 0.1 mM, NaF failed to affect most of these variables, except for a decrease in 45Ca net uptake. Much higher concentrations of NaF (e.g. 5.0 mM) were required to cause inhibition of the metabolic, ionic, biosynthetic and secretory responses of the islets to nutrient secretagogues. Yet, even at this high concentration, NaF failed to lower the islet content in tritiated A2P3 and A2P4 in islets prelabelled with [2,8-3H]adenosine and failed to prevent the glucose-ind...
The metabolism of alpha-D-glucose pentaacetate and its positive insulinotropic action in isolated... more The metabolism of alpha-D-glucose pentaacetate and its positive insulinotropic action in isolated rat pancreatic islets are both unexpectedly resistant to D-mannoheptulose, as judged from experiments conducted over 90-120 min incubation. In the present study, the possible effects of the heptose upon the immediate cationic and secretory response to the ester were investigated in perifused islets prelabeled with either 86Rb or 45Ca. At a 10 mM concentration, sufficient to abolish the inhibitory action of unesterified D-glucose upon 86Rb outflow, D-mannoheptulose failed to suppress the decrease in 86Rb outflow and increase in 45Ca efflux caused by alpha-D-glucose pentaacetate at normal extracellular Ca2+ concentration and also failed to prevent the decrease in both 45Ca and insulin release provoked by the ester in the absence of extracellular Ca2+. The sole obvious effect of the heptose was to change the early peak-shaped positive secretory response to alpha-D-glucose pentaacetate to a...
The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose... more The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5...
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Papers by Hassan Jijakli