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PubMed1982

Glucose-induced accumulation of fructose-2,6-bisphosphate in pancreatic islets.

Rat islets contain the acid-labile activator of phosphofructokinase, fructose-2,6-bisphosphate. The islet content in activator is higher in islets exposed to glucose (16.7 mM) than in islets deprived of glucose. The islets display fructose-6-phosphate, 2-kinase activity with a Km for fructose-6-phosphate close to 0.08 mM. Glucose fails to affect the activity of this enzyme. It is proposed that the effect of glucose to increase the islet content of fructose-2,6-bisphosphate is attributable, in part at least, to the glucose-induced increase in the concentration of fructose-6-phosphate in the islet cells.

Animals↗
PubMed1984

Tolbutamide stimulates fructose-2, 6-bisphosphate formation in perfused rat liver.

Effect of tolbutamide on liver fructose-2,6-bisphosphate (F-2,6-P2) was examined in isolated perfused rat liver in situ with a flow-through method. Tolbutamide (1 mM) gradually increased liver F-2,6-P2 level from 7.4 +/- 1.6 to 21.2 +/- 1.6 pmol/mg wet wt for 20 min perfusion. The increase of liver F-2,6-P2 induced by tolbutamide was dose dependent and was significantly observed at 10 min perfusion. The maximum plateau level of F-2,6-P2 induced by 16.7 mM glucose was further increased with 1 mM tolbutamide. Glucagon (10(-11) M) decreased the elevated level induced by 16.7 mM glucose, but this effect was completely inhibited with 2 mM tolbutamide. Cyclic AMP level of the liver throughout the perfusion with tolbutamide did not change. Carboxytolbutamide or gliclazide perfusion did not change significantly the liver F-2,6-P2 level; however, the results suggest that tolbutamide may increase the liver F-2,6-P2 level by affecting the phosphorylation state of fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase through cyclic AMP-dependent protein kinase, resulting in the stimulation of glycolysis and the inhibition of gluconeogenesis in the liver. Thus, the extrapancreatic action and the mechanism of action of different sulfonylureas may differ.

Animals↗
PubMed1985

Regulatory role of fructose-2,6-bisphosphate in pancreatic islet glucose metabolism remains unsettled.

Fructose-2,6-P2 was measured in perifused, isolated rat pancreatic islets. Fructose-2,6-P2 was present in pancreatic islets at low levels approximately equal to fructose-2,6-P2 content of liver from fasted rats. In islets perifused with glucose at physiologic concentrations, fructose-2,6-P2 was increased from 0.8 microM in the presence of 5.5 mM glucose to 1.0 microM at 10 mM glucose and 1.3 microM at 16.7 mM glucose, but did not increase further at higher glucose concentration. Therefore, only modest increases in the phosphofructokinase-1 activator, fructose-2,6-P2, occur at glucose concentrations stimulating insulin secretion.

Animals↗
PubMed1986

Chlorpropamide raises fructose-2,6-bisphosphate concentration and inhibits gluconeogenesis in isolated rat hepatocytes.

The addition of chlorpropamide to hepatocytes isolated from fed rats raised the cellular concentration of fructose-2,6-bisphosphate (F-2,6-P2), a regulatory metabolite that plays a relevant role in the control of hepatic glucose metabolism. The effect of chlorpropamide was dose dependent; a statistically significant increase was already seen at 0.2 mM of the sulfonylurea. The accumulation of F-2,6-P2 caused by chlorpropamide (1 mM) was parallel to the stimulation of L-lactate production (36.6 +/- 4.8 versus 26.1 +/- 2.6 mumol of lactate/g of cells X 20 min; N = 5, P less than 0.05) and to the inhibition of gluconeogenesis (0.57 +/- 0.1 versus 0.94 +/- 0.09 mumol of [U-14C]pyruvate converted to glucose/g of cells X 20 min; N = 5, P less than 0.05). In addition, chlorpropamide enhanced the inhibitory action evoked by insulin on glucagon-stimulated gluconeogenesis. This combined effect of chlorpropamide and insulin seems to be correlated with the synergistic accumulation of F-2,6-P2 provoked by the simultaneous action of these two agents on glucagon-treated hepatocytes. Finally, neither 6-phosphofructo-2-kinase activity nor hepatocyte cyclic AMP levels were significantly changed by the presence of the sulfonylurea in the incubation medium. Our results support the concept that chlorpropamide, by a cyclic AMP-independent mechanism, increases the hepatic content of F-2,6-P2 and, in this way, enhances the glycolytic flux and inhibits glucose output by the liver.

Animals↗
PubMed1986

Postreceptor myocardial metabolic defect in a rat model of non-insulin-dependent diabetes mellitus.

Hearts isolated from non-insulin-dependent diabetic rats were found to exhibit reduced rates of basal and insulin-stimulated glucose metabolism. Since tissue levels of fructose 1,6-bisphosphate are significantly reduced in the diabetic heart, it was concluded that phosphofructokinase may be inhibited. However, neither glycogen nor glucose 6-phosphate accumulated in the myocyte, indicating that the phosphofructokinase reaction was not a bottleneck diverting substrate away from glycolysis. The other major factor contributing to decreased glycolytic flux in the diabetic heart is the impairment in glucose transport. Both basal and insulin-stimulated transport of 3-O-methyl-D-glucose was 30% less in the diabetic heart. While insulin sensitivity was unaltered in the diabetic rat, insulin responsiveness was decreased, indicating that the impairment in insulin-stimulated hexose transport was caused by a post-receptor defect. The net result of these abnormalities in glucose metabolism is a significant reduction in the rate of ATP synthesis by the diabetic heart.

3-O-Methylglucose↗
PubMed1988

Control of glucose metabolism in pancreatic beta-cells by glucokinase, hexokinase, and phosphofructokinase. Model study with cell lines derived from beta-cells.

Glucose usage by soluble fractions of cell extracts from two insulin-producing cell lines, RINm5F and HIT, was investigated. Analysis of enzyme activities indicated that glucose phosphorylation and phosphofructokinase are likely to be the rate-limiting steps of glycolysis in both RINm5F and HIT cell extracts. RINm5F extracts, which lack glucokinase, exhibited relatively flat concentration-dependency curves of glucose usage and showed substantial inhibition of hexokinase. HIT cell extracts, which contain glucokinase but lack hexokinase, exhibited sigmoidal concentration-dependency curves of glucose usage, reflecting almost fully expressed glucokinase activity. A reconstituted system prepared from RINm5F and HIT cell extracts exhibited a composite concentration-dependency curve of glucose usage and showed substantial inhibition of hexokinase and almost fully expressed glucokinase. However, conditions that activate phosphofructokinase, such as addition of ammonium sulfate or fructose 2,6-bisphosphate or alkalization, removed the inhibition of hexokinase without noticeably affecting the glucokinase component of usage. Results obtained with a reconstituted system containing RINm5F cell extract and purified glucokinase were consistent with these findings. The data presented here indicate that this reconstituted cell-free system serves as a valid model for the study of aspects of glycolytic control in the islet. This model illustrates the preeminent role of glucokinase in the control of glycolysis, consistent with its glucose-sensor function in the islet. In addition, these studies help to define the contribution of phosphofructokinase to the control of glycolysis and the mechanism whereby changes in phosphofructokinase activity could modulate, via changes in the glucose 6-phosphate concentration, the activity of hexokinase and hence the net glycolytic flux.

Ammonium Sulfate↗
PubMed1991

Activation by vanadate of glycolysis in hepatocytes from diabetic rats.

In hepatocytes from starved streptozocin-induced diabetic rats, vanadate increases the glycolytic flux because it raises the levels of fructose-2,6-bisphosphate (Fru-2,6-P2), the main regulatory metabolite of this pathway. This effect of vanadate on Fru-2,6-P2 levels is time and dose dependent, and it remains in cells incubated in a calcium-depleted medium. Vanadate is also able to counteract the decrease on Fru-2,6-P2 levels produced by glucagon, colforsin, or exogenous cAMP. However, vanadate does not modify 6-phosphofructo-2-kinase and pyruvate kinase activities, but it does counteract the inactivation of these enzymes induced by glucagon. Likewise, Fru-2,6-P2ase activity is also not affected by vanadate. In addition, vanadate is able to increase the production of both lactate and CO2 in hepatocytes from streptozocin-induced diabetic rats incubated in the presence of glucose in the medium. Vanadate behaves as a glycolytic effector in these cells, and this effect may be related to its ability to normalize blood glucose levels in diabetic animals.

Animals↗
PubMed1994

cAMP-independent synergistic effects of insulin and dexamethasone on fructose 2,6-bisphosphate metabolism in H4IIE cells.

Hormonal regulation of fructose 2,6-bisphosphate (Fru-2,6-P2) content was studied in H4IIE cells. These cells were found to be very sensitive to physiological concentrations of insulin. Addition of either insulin or dexamethasone alone increased Fru-2,6-P2 in a time- and dose-dependent manner, and the maximal effect of the hormones was seen at 1 h. Neither hormone had any measurable effect on cAMP levels. The effect of addition of both insulin and dexamethasone on Fru-2,6-P2 was synergistic. Insulin, but not dexamethasone, rapidly increased 6-phosphofructo-2-kinase (6PF-2-K) activity by causing dephosphorylation of the enzyme as judged by a decrease in the Km for fructose-6-phosphate. Addition of both hormones also resulted in a synergistic 10-fold increase in enzyme protein as measured by kinase activity and phosphoenzyme formation. Dexamethasone increased liver 6PF-2-K/Fru-2,6-P2 mRNA abundance by 10- to 12-fold as measured by a ribonuclease protection assay, and insulin increased it by only 4-fold. Effects were observed as early as 1 h after hormone addition, but addition of both hormones together showed no synergy. We conclude that the synergistic effects of insulin and dexamethasone on Fru-2,6-P2 content are mediated by a combination of stimulation of expression of the bifunctional enzyme gene by both hormones and insulin-induced modulation of the activation state of the bifunctional enzyme, both of which are mediated by cAMP-independent mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Phosphofructokinase isozymes in pancreatic islets and clonal beta-cells (INS-1).

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1997

Oscillations in oxygen consumption by permeabilized clonal pancreatic beta-cells (HIT) incubated in an oscillatory glycolyzing muscle extract: roles of free Ca2+, substrates, and the ATP/ADP ratio.

To determine whether oscillations in glycolysis could underlie the oscillations in O2 consumption observed in intact islets, we evaluated the capacity of an islet extract to exhibit spontaneous oscillations in glycolysis. When a cell-free extract obtained from approximately 1,000 islets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spontaneous oscillations in islet extracts, we bathed permeabilized clonal beta-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of alpha-glycerophosphate and pyruvate and induces oscillations in free Ca2+ and the ATP/ADP ratio. This preparation was used to investigate whether changes in Ca2+ and possibly alpha-glycerophosphate or pyruvate supply could underlie observed oscillations in O2 consumption and explain coordination between cytosolic and mitochondrial metabolism. We found that oscillations of O2 consumption and Ca2+ of a similar period were induced. Removal of medium Ca2+ with EGTA did not prevent the oscillations in O2 consumption nor were they greatly affected by the substantial rise in medium Ca2+ on treatment with thapsigargin to inhibit sequestration into the endoplasmic reticulum. The 02 oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or alpha-glycerophosphate. However, they were lost on addition of fructose-2,6-P2 at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased 02 consumption and also prevented 02 oscillations. These results suggest that the changes in respiration reflected in the 02 oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of 02 consumption in the pancreatic beta-cell.

Adenosine Diphosphate↗
PubMed1997

Oscillations in activities of enzymes in pancreatic islet subcellular fractions induced by physiological concentrations of effectors.

Glucose, the most potent insulin secretagogue, stimulates insulin secretion by aerobic glycolysis, but other secretagogues stimulate insulin release exclusively by mitochondrial metabolism. It is well known that in the intact pancreatic beta-cell, either kind of secretagogue can induce oscillations in metabolism (e.g., glycolysis, ATP/ADP, NAD(P)/NAD(P)H ratios) that occur with a periodicity similar to oscillations in membrane electrical potential and insulin secretion. In this study, pancreatic islet cytosol or mitochondrial fractions were incubated in the presence of physiological concentrations of substrates. Repeated additions of physiological effectors caused oscillations in the activities of the three enzymes studied. Succinate dehydrogenase activity in islet mitochondrial extracts was made to oscillate by adding oxaloacetate (5 micromol/l) to inhibit the enzyme. The enzyme was reactivated by adding acetyl-CoA (3 micromol/l), which combines with oxaloacetate in the citrate synthase reaction and lowers the concentration of oxaloacetate, thus beginning another oscillation. Pyruvate kinase activity was made to oscillate by adding fructose bisphosphate (10 micromol/l). Fructose bisphosphate was degraded to triose phosphates fairly rapidly, and, as it was degraded, there was a parallel decrease in pyruvate kinase activity. The enzyme was reactivated and made to oscillate with subsequent additions of fructose bisphosphate. The mitochondrial glycerol phosphate dehydrogenase was made to oscillate by adding EGTA to chelate calcium, which activates the enzyme. When the concentration of free calcium was raised to >0.1 micromol/l by adding more calcium, the activity of the enzyme increased. Repeated additions of chelator and calcium caused the enzyme activity to oscillate. The results with these three enzymes and physiological concentrations of naturally occurring effectors raise the possibility that the activities of not only these enzymes but of numerous enzymes oscillate in vivo in response to levels of allosteric effectors and substrates. If this is the case, pacemaker activity may result from complex effects distributed across multiple regulatory sites in both the cytosol and mitochondria, rather than from a single enzyme acting as a primary pacemaker.

Acetyl Coenzyme A↗
PubMed1997

Pyruvate improves deleterious effects of high glucose on activation of pentose phosphate pathway and glutathione redox cycle in endothelial cells.

In our previous study (Diabetes 44:520-526, 1995), endothelial cells cultured in high glucose condition showed impairment of an oxidant-induced activation of the pentose phosphate pathway (PPP) and a reduced supply of NADPH to the glutathione redox cycle. To gain insight into the mechanisms of this impairment, the protective effect of pyruvate was studied in human umbilical vein endothelial cells cultured in either 5.5 mmol/l glucose (normal glucose [NG] condition) or 33 mmol/l glucose (high glucose [HG] condition). Through pretreatment of cells with 0.2 mmol/l pyruvate for 5-7 days in the HG condition, glucose oxidation through the PPP and total cellular NADPH content in the presence of 0.2 mmol/l H2O2 were increased by 54 (P < 0.05) and 34%, respectively, and glutathione-dependent degradation of H2O2 in HG cells was enhanced by 41% (P < 0.01), when compared with those cells to which pyruvate was not added. The addition of pyruvate significantly reduced the fructose 1,6-bisphosphate (FDP) content and free cytoplasmic NADH/NAD ratio, estimated by increased pyruvate/lactate ratio in NG and HG cells exposed to H2O2. Furthermore, the addition of pyruvate also showed a 46% reduction (P < 0.01) of endothelial cell damage induced by H2O2 in HG cells. These results indicate that abnormalities in PPP activation and glutathione redox cycle activity induced by H2O2 in HG cells are compensated, and that the accentuated reductive stress is improved by an addition of pyruvate. These pyruvate effects are associated with protection against an oxidant-induced endothelial cell injury in the high glucose condition.

Adenosine Triphosphate↗
PubMed1990

Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis. From metabolites to molecular genetics.

Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes. Regulation by cAMP-dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P2ase). The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P2ase. The bifunctional enzyme is a homodimer whose activities are regulated by cAMP-dependent protein kinase-catalyzed phosphorylation at a single NH2-terminal seryl residue/subunit, which results in activation of the Fru-2,6-P2ase and inhibition of the PF-1-K reactions. Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P2 levels. 6PF-2-K/Fru-2,6-P2ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Pyruvate kinase is regulated by both phosphorylation and allosteric effectors. Fru-1,6-P2, an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P2. Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P2 substrate cycles. The Fru-2,6-P2 system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P2ase gene expression. Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector. cAMP prevents the effects of glucocorticoids and insulin. Although Fru-2,6-P2 plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal. Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P2ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression. The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene. The muscle form differs from the liver form only at the NH2-terminal and does not have a cAMP-dependent protein kinase phosphorylation site. The hepatic enzyme subunit consists of 470 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1978

Muscle metabolism during and after strenuous intermittent running.

Muscle and blood metabolites, plasma insulin and cyclic adenosine 3',5'-monophosphate (cAMP) levels were investigated in five male runners before and after strenuous intermittent running exercise of short duration. Immediately after the exercise, the mean muscle creatine phosphate level (CrP) had fallen by 74% (P less than 0.02) and 30 min later the initial level was regained in only one subject. Other immediate results were increases in mean muscle lactate (460%, P less than 0.005), glucose (130%), glucose-6-phosphate (G6P, 320%) and fructose-1,6-diphosphate (FDP, 32%). Muscle ATP and glycogen concentration had decreased by 31 and 23% (P less than 0.05), respectively. However, ATP, glucose, G6P and FDP changes were not significant owing to the great individual variation. This may have been due to the different training programmes of the runners. Immediately after the exercise mean plasma insulin was 210% (P less than 0.01), blood glucose 71% (P less than 0.005) and plasma cAMP concentration 260% (P less than 0.01) higher than the pre-exercise values. After running urinary excretion of cAMP was 29% higher than before the exercise. It is concluded that exhaustive, short-term exercise activates the liver adenylate cyclase system so giving rise to an increased level of blood glucose, which is an important source of energy during this type of exercise.

Adenosine Triphosphate↗
PubMed1981

Phosphate metabolite regulation of spectrin interactions.

The deformability of the erythrocyte membrane is believed to depend upon component interactions in the spectrin, actin and band 4.1 complex. Phosphate metabolites, such as 2,3-diphosphoglycerate (2,3 DPG) will dissociate spectrin from actin and band 4.1. This dissociation by 2,3 DPG is highly pH dependent but does not involve divalent cations, 2,3 DPG hydrolysis or spectrin dephosphorylation. In intact erythrocytes the concentrations of 2,3 DPG and the lipid, triphosphatidyl inositol, are sufficient to cause increased labilization in the spectrin, actin and band 4.1 network.

2,3-Diphosphoglycerate↗
PubMed1980

Pyruvate kinase deficiency hemolytic anemia: enzymatic characterization studies in twelve patients.

Erythrocyte pyruvate kinase from twelve patients with hereditary erythrocyte pyruvate kinase (PK) deficiency was investigated according to the recommended methods for the characterization of red cell pyruvate kinase variants. Family members were also studied. Abnormalities were frequently noted in the affinity for the substrate, phosphoenolpyruvate; allosteric activator, fructose-1,6-diphosphate; allosteric inhibitor, ATP; and also in heat stability and electrophoretic pattern. Several different PK variants were identified. Polyacrylamide gel electrophoresis revealed the presence of immature activity bands in the red cells of some patients. These bands presumably represent residuals of isozymes produced during the evolution of erythrocyte PK, and may occur as a compensatory mechanism for the defective isozyme.

Adenosine Triphosphate↗
PubMed1991

Erythrocyte fructose 2,6-bisphosphate content in congenital hemolytic anemias.

We have investigated the levels of fructose 2,6-bisphosphate and its synthesizing enzyme 6-phosphofructo-2-kinase in red blood cells from different congenital anemias. Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity are markedly influenced by the number of reticulocytes in all the cases studied with the exception of homozygous pyruvate kinase deficiency, where no correlation was observed with the percentage of reticulocytes.

Anemia, Hemolytic, Congenital↗