Mannose inhibits Arabidopsis germination via a hexokinase-mediated step. (1/54)

Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion.  (+info)

Metabolic regulation, activity state, and intracellular binding of glucokinase in insulin-secreting cells. (2/54)

Regulation of glucose-induced insulin secretion is crucially dependent on glucokinase function in pancreatic beta-cells. Glucokinase mRNA expression was metabolically regulated allowing continuous translation into enzyme protein. Glucokinase enzyme activity in the beta-cell was exclusively regulated by glucose. Using a selective permeabilization technique, different intracellular activity states of the glucokinase enzyme in bioengineered glucokinase-overexpressing RINm5F tissue culture cells were observed. These results could be confirmed in analogous experiments with dispersed islet cells. A diffusible glucokinase fraction with high enzyme activity could be distinguished from an intracellularly bound fraction with low activity. Glucose induced a significant long-term increase of the active glucokinase fraction. This effect was accomplished through the release of glucokinase enzyme protein from an intracellular binding site of protein character. The inhibitory function of this protein factor was abolished through proteolytic digestion or heat inactivation. Northern blot analyses revealed that this binding protein was not identical to the well-known liver glucokinase regulatory protein. This hitherto unknown new protein factor may have the function of a glucokinase regulatory protein in the pancreatic beta-cell, which may regulate glucokinase enzyme activity in a glucose-dependent manner.  (+info)

Cellular origin of hexokinase in pancreatic islets. (3/54)

Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low K(m) hexokinases (HK) exhibit a leftward shift of the normal dose-response curve for glucose-induced insulin release. Furthermore, HK catalyzes roughly 50% of total glucose phosphorylation measured in extracts from freshly isolated rodent islets, suggesting that HK participates in the process of glucose sensing in beta cells. We previously observed that HK activity represents 20% of total glucose phosphorylation in purified rat beta cell preparations and that HK is not homogenously distributed over these cells. The present study provides several arguments for the idea that HK detected in freshly isolated rat islets or islet cell preparations originates mainly from contaminating exocrine cells. First, reverse transcriptase-polymerase chain reaction using isoform-specific primers allowed detection of hexokinase I and IV mRNA in rat beta cells, whereas the messenger levels encoding the hexokinase II and III isoforms were undetectably low. However, immunoblots indicated that hexokinase I protein was 10-fold more abundant in freshly isolated islets and flow-sorted exocrine cells than in purified rat beta cell preparations. Second, comparison of HK activity in the different pancreatic cell types resulted in 15-25-fold higher values in exocrine than in endocrine cells (acinar cells: 21 +/- 3 pmol of glucose 6-phosphate formed/h/ng of DNA; duct cells: 30 +/- 8 pmol/h/ng of DNA; islet beta cells: 1.2 +/- 0.2 pmol/h/ng DNA; alpha cells: 0.9 +/- 0.4 pmol/h/ng of DNA). Since freshly purified beta cell preparations contain 3 +/- 1% exocrine cells, at least 50% of their HK activity can be accounted for by exocrine contamination. Third, after 5 days of culture of purified islet beta cells, both HK activity and the proportion of exocrine cells decreased by more than 1 order of magnitude, while the ratio of glucokinase over hexokinase activity increased more than 10-fold. Finally, preincubating the cells with 50 mmol/liter 2-deoxyglucose did not affect glucose stimulation of insulin biosynthesis and release. In conclusion, the observation that pancreatic exocrine cells are responsible for a major part of HK activity in islet cell preparations cautions against the use of HK measurements in islet extracts in the study of these enzymes in glucose sensing by pancreatic beta cells.  (+info)

Pancreatic islet cells: effects of monosaccharides, glycolytic intermediates and metabolic inhibitors on membrane potential and electrical activity. (4/54)

1. The effects of monosaccharides, glycolytic intermediates, metabolic inhibitors and anxia, have been studied on the membrane electrical activity of mouse pancreatic islet cells in vitro using a single intracellular micro-electrode for both voltage recording and current injection. 2. In addition to D-glucose (28mM), D-mannose (16-6mM), and L-leucin (10mM), the substances D-glyceraldehyde (11mM), and acetoacetate (20 mM), induced action potentials in islet cells but other glucos analogues and metabolic intermediates including L-glucose dod not. 3. Mannoheptulose 20 mM), but not D-galactose or 2-deoxy-D-glucose, antagonized the electrical activity induced in islet cells by D-glucose, 28mM. Prior treatment of the cells with mannoheptulose caused them to hyperpolarize and completely prevented the appearance of electrical activity on subsequent exposure to D-glucose. 4. Electrical activity induced by D0glucose 28mM, was progressively inhibited by phloridzin, 10mM, if the cells were exposed to D-glucose and inhibitor simultaneously, and abolished on pretreatment with inhibitor for 30-60 min. Phloridzin also caused depolarization of the islet cells which was independent of extracellular glucose. 5. Anoxia completely blocked the electrical activity induced by glucose but not that evoked by D-glyceraldehyde, L-leucine, tolbutamide or glibenclamide. 6. Iodoacetic acid, 5 mM, rapidly blocked glucose-induced electrical activity whilst that elicited by tolbutamide was relatively resistant to inhibition. 7. The nature and possible location of the glucoreceptor in pancreatic islet cells is discussed in relation to the origin and functional significance of glucose-induced electrical activity and insulin secretion.  (+info)

Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose. (5/54)

We have found that preexposure to an elevated concentration of glucose reversibly induces an enhancement of basal insulin release from rat pancreatic islets dependent on glucose metabolism. This basal insulin release augmented by priming was not suppressed by reduction of the intracellular ATP or Ca(2+) concentration, because even in the absence of ATP at low Ca(2+), the augmentation was not abolished from primed electrically permeabilized islets. Moreover, it was not inhibited by an alpha-adrenergic antagonist, clonidine. A threshold level of GTP is required to induce these effects, because together with adenine, mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely abolished the enhancement of basal insulin release due to the glucose-induced priming without affecting the glucose-induced increment in ATP content and ATP-to-ADP ratio. In addition, a GDP analog significantly suppressed the enhanced insulin release due to priming from permeabilized islets in the absence of ATP at low Ca(2+), suggesting that the GTP-sensitive site may play a role in the augmentation of basal insulin release due to the glucose-induced priming effect.  (+info)

Assessment of islet beta-cell mass in isolated rat pancreases perfused with D-[(3)H]mannoheptulose. (6/54)

D-mannoheptulose is apparently transported into cells mainly at the intervention of GLUT-2 and hence was recently proposed as a tool to label preferentially insulin-producing beta-cells in the pancreatic gland. The validity of such a proposal was investigated in the present study conducted in isolated perfused pancreatic glands from control and streptozotocin-induced diabetic rats. After a 30-min equilibration period, D-[(3)H]mannoheptulose (0.1 mM) and [U-(14)C]sucrose (0.5 mM) were infused for 15 min in the presence of 30 mM D-glucose. The pancreatic glands were then perfused for 10 min with a nonradioactive medium during and after administration of cytochalasin B (0.02 mM). Under these experimental conditions, the intracellular distribution space of D-[(3)H]mannoheptulose averaged 5.42 +/- 0.75 nl/mg in control animals, whereas it failed to be significantly different from zero in the streptozotocin rats. The present procedure may thus allow the assessment of the relative contribution of islet beta-cells to the total mass of the pancreatic gland.  (+info)

Correlation between GABA release from rat islet beta-cells and their metabolic state. (7/54)

Pancreatic beta-cells express glutamate decarboxylase (GAD), which is responsible for the production and release of gamma-aminobutyric acid (GABA). Over a 24-h culture period, total GABA release by purified rat beta-cells is eightfold higher than the cellular GABA content and can thus be used as an index of cellular GAD activity. GABA release is 40% reduced by glucose (58 pmol/10(3) cells at 10 mM glucose vs. 94 pmol at 3 mM glucose, P < 0.05). This suppressive effect of glucose was not observed when glucose metabolism was blocked by mannoheptulose or 2,4-dinitrophenol; it was amplified when ATP-dependent beta-cell activities were inhibited by addition of diazoxide, verapamil, or cycloheximide or by reduction of extracellular calcium levels; it was counteracted when beta-cell functions were activated by nonmetabolized agents, such as glibenclamide, IBMX, glucagon, or glucacon-like peptide-1 (GLP-1), which are known to stimulate calcium-dependent activities, such as hormone release and calcium-dependent ATPases. These observations suggest that GABA release from beta-cells varies with the balance between ATP-producing and ATP-consuming activities in the cells. Less GABA is released in conditions of elevated glucose metabolism, and hence ATP production, but this effect is counteracted by ATP-dependent activities. The notion that increased cytoplasmic ATP levels can suppress GAD activity in beta-cells, and hence GABA production and release, is compatible with previous findings on ATP suppression of brain GAD activity.  (+info)

Role of glucose 6-phosphate in the translocation of glycogen synthase in rat hepatocytes. (8/54)

Incubation of rat hepatocytes with glucose induces the translocation of glycogen synthase from soluble fractions to fractions which sediment at 10,000 g. Incubation of the cells with fructose, galactose, 2-deoxyglucose or 5-thioglucose, which activate glycogen synthase, also resulted in the translocation of the enzyme, whereas 3-O-methylglucose, 6-deoxyglucose and 1,5-anhydroglucitol, which do not cause the activation of the enzyme, were ineffective. Adenosine and carbonyl cyanide m-chlorophenylhydrazone, although activating glycogen synthase, did not induce its translocation. Mannoheptulose, which inhibits glucose phosphorylation, impaired the translocation of glycogen synthase induced by glucose. Furthermore, the extent of the translocation of the enzyme triggered by glucose and other sugars showed a high positive correlation with the intracellular concentration of glucose 6-phosphate. Microcystin, which blocks the activation of glycogen synthase by glucose, but not the accumulation of glucose 6-phosphate, did not affect the translocation of the enzyme. These results indicate that glucose 6-phosphate plays a role in the translocation of glycogen synthase in rat hepatocytes.  (+info)

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