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Kosaku Uyeda - One of the best experts on this subject based on the ideXlab platform.
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increased hepatic Fructose 2 6 bisphosphate after an oral glucose load does not affect gluconeogenesis
Journal of Biological Chemistry, 2003Co-Authors: Eunsook S Jin, Kosaku Uyeda, Takumi Kawaguchi, Shawn C Burgess, Craig R Malloy, Dean A SherryAbstract:Abstract The generally accepted metabolic concept that Fructose 2,6-bisphosphate (Fru-2,6-P2) inhibits gluconeogenesis by directly inhibiting Fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P2 in intact animals, a novel NMR method was developed using [U-13C]glucose and 2H2O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P2 increased ∼10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached ∼50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching ∼30 μmol/g at 3 h. These data demonstrate that Fru-2,6-P2 does not inhibit gluconeogenesis significantly in vivo.
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regulation of energy metabolism in macrophages during hypoxia roles of Fructose 2 6 bisphosphate and ribose 1 5 bisphosphate
Journal of Biological Chemistry, 2001Co-Authors: Takumi Kawaguchi, Richard L Veech, Kosaku UyedaAbstract:Macrophages can adapt to the absence of oxygen by switching to anaerobic glycolysis. In this study, we investigated (a) the roles of Fructose 2,6-bisphosphate (Fru-2,6-P2) and ribose 1,5-bisphosphate (Rib-1,5-P2), potent activators of phosphofructokinase, (b) the enzymes responsible for the synthesis of Rib-1,5-P2, and (c) the mechanisms of regulation of these enzymes in H36.12j macrophages during the initial phase of hypoxia. Within 1 min after initiating hypoxia, glycolysis was activated through activation of phosphofructokinase. Over the same period, Fru-2,6-P2 decreased 50% and recovered completely upon reoxygenation. Similar changes in cAMP levels were observed. In contrast, the Rib-1,5-P2 concentration rapidly increased to a maximum level of 8.0 +/- 0.9 nmol/g cell 30 s after hypoxia. Thus, Rib-1,5-P2 was the major factor increasing the rate of glycolysis during the initial phase of hypoxia. Moreover, we found that Rib-1,5-P2 was synthesized by two steps: the ribose-phosphate pyrophosphokinase (5-phosphoribosyl-1-pyrophosphate synthetase; PRPP synthetase) reaction (EC ) catalyzing the reaction, Rib-5-P + ATP --> PRPP + AMP and a new enzyme, "PRPP pyrophosphatase" catalyzing the reaction, PRPP --> Rib-1,5-P2 + P(i). Both PRPP synthetase and PRPP pyrophosphatase were significantly activated 30 s after hypoxia. Pretreatment with 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine and calphostin C prevented the activation of ribose PRPP synthetase and PRPP pyrophosphatase as well as increase in Rib-1,5-P2 and activation of phosphofructokinase 30 s after hypoxia. These data suggest that the activation of the above enzymes was mediated by protein kinase C acting via activation of phosphatidylinositol specific phospholipase C in the macrophages during hypoxia.
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the crystal structure of the bifunctional enzyme 6 phosphofructo 2 kinase Fructose 2 6 bisphosphatase reveals distinct domain homologies
Structure, 1996Co-Authors: Charles A Hasemann, Kosaku Uyeda, Eva S Istvan, Johann DeisenhoferAbstract:Abstract Background Glucose homeostasis is maintained by the processes of glycolysis and gluconeogenesis. The importance of these pathways is demonstrated by the severe and life threatening effects observed in various forms of diabetes. The bifunctional enzyme 6-phosphofructo-2-kinase/Fructose-2,6-bisphosphatase catalyzes both the synthesis and degradation of Fructose-2,6-bisphosphate, a potent regulator of glycolysis. Thus this bifunctional enzyme plays an indirect yet key role in the regulation of glucose metabolism. Results We have determined the 2.0 a crystal structure of the rat testis isozyme of this bifunctional enzyme. The enzyme is a homodimer of 55 kDa subunits arranged in a head-to-head fashion, with each monomer consisting of independent kinase and phosphatase domains. The location of ATP γ S and inorganic phosphate in the kinase and phosphatase domains, respectively, allow us to locate and describe the active sites of both domains. Conclusions The kinase domain is clearly related to the superfamily of mononucleotide binding proteins, with a particularly close relationship to the adenylate kinases and the nucleotide-binding portion of the G proteins. This is in disagreement with the broad speculation that this domain would resemble phosphofructokinase. The phosphatase domain is structurally related to a family of proteins which includes the cofactor independent phosphoglycerate mutases and acid phosphatases.
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glucose stimulated synthesis of Fructose 2 6 bisphosphate in rat liver dephosphorylation of Fructose 6 phosphate 2 kinase Fructose 2 6 bisphosphatase and activation by a sugar phosphate
Journal of Biological Chemistry, 1994Co-Authors: Motonobu Nishimura, Sergei Fedorov, Kosaku UyedaAbstract:The effect of glucose on hepatic Fructose (Fru) 2,6-P2 in starved rats was investigated. When livers were perfused with high glucose (40 mM), hexose-P in the liver increased immediately reaching the maximum within in 2 min, but Fru 2,6-P2 after a lag period of 4 min increased linearly. The activation of Fru 6-P,2-kinase and inactivation of Fru 2,6-Pase also showed a similar lag period. Determination of the phosphate contents of the bifunctional enzyme after 10 min of glucose perfusion revealed that 90% of the enzyme was in the dephospho form while only 10% of the control liver enzyme was dephosphorylated. Comparison of crude extracts of liver perfused with either high glucose or normal glucose (5.6 mM) showed that high glucose livers contained 50% higher protein phosphatase activity, which dephosphorylated the bifunctional enzyme. Subcellular fractionation of the extract showed that activation of the protein phosphatase occurred in the cytosol. Desalting of the cytosolic fraction resulted in a 50% loss of the protein phosphatase activity. The low molecular weight activator in the cytosol was isolated, and by various chemical and enzymatic methods it was identified as xylulose 5-P. The activation of protein phosphatase by xylulose 5-P showed a highly sigmoidal saturation curve. The rate of formation of xylulose 5-P in the perfused liver showed a lag period of approximately 2 min, and after 4 min its concentration reached 10 microM, the minimum concentration necessary for the activation of the protein phosphatase. We conclude that the mechanism of glucose-induced Fru 2,6-P2 synthesis was not due to increased Fru 6-P as generally thought but occurred as a result of dephosphorylation of Fru 6-P,2-kinase:Fru 2,6-Pase. Moreover, the dephosphorylation was enhanced by increased xylulose 5-P, which activated a specific protein phosphatase. The results suggest a mechanism for coordinated regulation of glycolysis and the pentose shunt pathway that is mediated by xylulose 5-P.
Louis Hue - One of the best experts on this subject based on the ideXlab platform.
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6 phosphofructo 2 kinase Fructose 2 6 bisphosphatase head to head with a bifunctional enzyme that controls glycolysis
Biochemical Journal, 2004Co-Authors: Mark H. Rider, Guy G Rousseau, Luc Bertrand, Didier Vertommen, Paul A M Michels, Louis HueAbstract:Fru-2,6-P2 (Fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (Fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.
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apoptosis induced by growth factor withdrawal in fibroblasts overproducing Fructose 2 6 bisphosphate
FEBS Letters, 1999Co-Authors: Paula Durante, Louis Hue, Marieagnes Gueuning, Martine I Darville, Guy G RousseauAbstract:Fructose 2,6-bisphosphate is a potent endogenous stimulator of glycolysis. A high aerobic glycolytic rate often correlates with increased cell proliferation. To investigate this relationship, we have produced clonal cell lines of Rat-1 fibroblasts that stably express transgenes coding for 6-phosphofructo-2-kinase, which catalyzes the synthesis of Fructose 2,6-bisphosphate, or for Fructose 2,6-bisphosphatase, which catalyzes its degradation. While serum deprivation in culture reduced the growth rate of control cells, it caused apoptosis in cells overproducing Fructose 2,6-bisphosphate. Apoptosis was inhibited by 5-amino-4-imidazolecarboxamide riboside, suggesting that 5'-AMP-activated protein kinase interferes with this phenomenon.
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Signaling pathway involved in the activation of heart 6-phosphofructo-2-kinase by insulin.
Journal of Biological Chemistry, 1996Co-Authors: Véronique Lefebvre, Mark H. Rider, Marie Claire Méchin, Marc P. Louckx, Louis HueAbstract:Incubation of isolated rat cardiomyocytes with insulin increased 2-deoxyglucose uptake, glycogen synthesis, and Fructose 2, 6-bisphosphate content. Half-maximal effects were obtained with 1-2 nM insulin. The insulin-induced increase in Fructose 2,6-bisphosphate content was preceded by a 2-3-fold activation of 6-phosphofructo-2-kinase, which was independent of glucose transport. Insulin activated phosphatidylinositol 3-kinase and p70 ribosomal S6 kinase (p70 S6 kinase), but had no significant effect on mitogen-activated protein kinase, although phorbol 12-myristate 13-acetate activated the latter. The effect of insulin on Fructose 2, 6-bisphosphate, 6-phosphofructo-2-kinase, and phosphatidylinositol 3-kinase was blocked by wortmannin. However, rapamycin, which inhibited p70 S6 kinase activation, and PD 98059, an inhibitor of the mitogen-activated protein kinase pathway, had no effect on the insulin-induced activation of 6-phosphofructo-2-kinase. Heart 6-phosphofructo-2-kinase can therefore be regarded as a glycolytic target of insulin. Its activation by insulin might be mediated by phosphatidylinositol 3-kinase.
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role of Fructose 2 6 bisphosphate in the control of heart glycolysis
Journal of Biological Chemistry, 1993Co-Authors: Christophe Depre, Mark H. Rider, Keith Veitch, Louis HueAbstract:The aim of this work was to study whether changes in Fructose 2,6-bisphosphate concentration are correlated with variations of the glycolytic flux in the isolated working rat heart. Glycolysis was stimulated to different extents by increasing the concentration of glucose, increasing the workload, or by the addition of insulin. The glycolytic flux was measured by the rate of detritiation of [2-3H]- and [3-3H]glucose. Under all the conditions tested, an increase in Fructose 2,6-bisphosphate content was observed. The glucose- or insulin-induced increase in Fructose 2,6-bisphosphate content was related to an increase in the concentration of Fructose 6-phosphate, the substrate of 6-phosphofructo-2-kinase. An increase in the workload correlated with a 50% decrease in the Km of 6-phosphofructo-2-kinase for Fructose 6-phosphate. Similar changes in Km have been observed when purified heart 6-phosphofructo-2-kinase was phosphorylated in vitro by the cyclic AMP-dependent protein kinase or by the calcium/calmodulin-dependent protein kinase. Since the concentration of cyclic AMP was not affected by increasing the workload, it is possible that the change in Km of 6-phosphofructo-2-kinase, which was found in hearts submitted to a high load, resulted from phosphorylation by calcium/calmodulin protein kinase; other possibilities are not excluded. Anoxia decreased the external work developed by the heart, stimulated glycolysis and glycogenolysis, but did not increase Fructose 2,6-bisphosphate.
Tom Hamborg Nielsen - One of the best experts on this subject based on the ideXlab platform.
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osmotic stress changes carbohydrate partitioning and Fructose 2 6 bisphosphate metabolism in barley leaves
Functional Plant Biology, 2005Co-Authors: Dorthe Villadsen, Jesper Henrik Rung, Tom Hamborg NielsenAbstract:Carbohydrate metabolism was investigated in barley leaves subjected to drought or osmotic stress induced by sorbitol incubation. Both drought and osmotic stress resulted in accumulation of hexoses, depletion of sucrose and starch, and 5–10-fold increase in the level of the regulatory metabolite Fructose-2,6-bisphosphate (Fru-2,6-P2). These changes were paralleled by an increased activity ratio of Fructose-6-phosphate,2-kinase / Fructose-2,6-bisphosphatase (F2KP). The drought-induced changes in carbohydrate content and Fru-2,6-P2 metabolism were reversed upon re-watering. This reveals a reversible mechanism for modification of the F2KP enzyme activity. This suggests that F2KP might be involved in altering carbohydrate metabolism during osmotic stress. However, labelling with [14C]-CO2 showed that sucrose synthesis was not inhibited, despite the increased Fru-2,6-P2 levels, and demonstrated that increased flux into the hexose pools probably derived from sucrose hydrolysis. Similar effects of osmotic stress were observed in leaf sections incubated in the dark, showing that the changes did not result from altered rates of photosynthesis. Metabolism of [14C]-sucrose in the dark also revealed increased flux into hexoses and reduced flux into starch in response to osmotic stress. The activities of a range of enzymes catalysing reactions of carbohydrate metabolism in general showed only a marginal decrease during osmotic stress. Therefore, the observed changes in metabolic flux do not rely on a change in the activity of the analysed enzymes. Fructose-2,6-bisphosphate metabolism responds strongly to drought stress and this response involves modification of the F2KP activity. However, the data also suggests that the sugar accumulation observed during osmotic stress is mainly regulated by another, as yet unidentified mechanism.
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Fructose 2 6 bisphosphate a traffic signal in plant metabolism
Trends in Plant Science, 2004Co-Authors: Tom Hamborg Nielsen, Jesper Henrik Rung, Dorthe VilladsenAbstract:Fructose-2,6-bisphosphate (Fru-2,6-P 2 ) regulates key reactions of the primary carbohydrate metabolism in all eukaryotes. In plants, Fru-2,6-P 2 coordinates the photosynthetic carbon flux into sucrose and starch biosynthesis. The use of transgenic plants has allowed the regulatory models to be tested by modifying the Fru-2,6-P 2 levels and the enzymes regulated by Fru-2,6-P 2 . Genes for the bifunctional plant enzyme that synthesizes and degrades Fru-2,6-P 2 have been isolated and molecular characterization has provided new insight into structure and molecular regulation of the enzyme. Advances in Fru-2,6-P 2 physiology and molecular biology are discussed. These advances have not only enlightened in vivo operation of Fru-2,6-P 2 but also revealed that the Fru-2,6-P 2 regulatory system is highly complex and interacts with other regulatory mechanisms.
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quantitative aspects of the in vivo regulation of pyrophosphate Fructose 6 phosphate 1 phosphotransferase by Fructose 2 6 bisphosphate
Plant Physiology, 1995Co-Authors: Tom Hamborg Nielsen, Bente WischmannAbstract:Pyrophosphate:Fructose-6-phosphate 1-phosphotransferase (PFP) was quantified in developing barley (Hordeum vulgare) leaves by immunostaining on western blots using a purified preparation of barley leaf PFP as standard. Fructose-2,6-bisphosphate (Fru-2,6-bisP) was quantified in the same tissues. Depending on age and tissue development, the concentration of PFP varied between 11 and 80 [mu]g PFP protein g-1 fresh weight, which corresponds to 0.09 to 0.65 nmol g-1 fresh weight of each of the [alpha] and [beta] PFP subunits. The level depends primarily on the maturity of the tissue. In the same tissues the concentration of Fru-2,6-bisP varied between 0.07 and 0.46 nmol g-1 fresh weight. Thus, the concentrations of PFP subunits and Fru-2,6-bisP were of the same order of magnitude. In young leaf tissues the concentration of PFP subunits may exceed the concentration of Fru-2,6-bisP. This means that the amount of Fru-2,6-bisP present will be too low to occupy all the allosteric binding sites on PFP even though the concentration of Fru-2,6-bisP exceeds the Ka(Fru-2,6-bisP) by several orders of magnitude. These results are discussed in relation to Fru-2,6-bisP as a regulator of enzyme activities under in vivo conditions.
Geoffrey D Holman - One of the best experts on this subject based on the ideXlab platform.
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d Fructose l sorbose interconversions access to 5 thio d Fructose and interaction with the d Fructose transporter glut5
Carbohydrate Research, 2001Co-Authors: Arnaud Tatibouet, Myriam Lefoix, Jonathan Nadolny, Olivier R Martin, Patrick Rollin, Jing Yang, Geoffrey D HolmanAbstract:Abstract Epimerisation and subsequent functionalization at C-5 of d -fructopyranose derivatives under Mitsunobu and Garegg's conditions provided efficient access to 5-thio- d -Fructose ( 2 ) as well as to 5-azido-5-deoxy-1,2- O -isopropylidene-β- d -fructopyranose ( 19 ), a known precursor to 2,5-deoxy-2,5-imino- d -mannitol ( 3 ). The interaction of 2 with the d -Fructose transporter GLUT5, was found to be weaker than that of d -Fructose, a result that suggests involvement of the ring oxygen atom in the recognition of d -Fructose by GLUT5.
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d-Fructose–l-sorbose interconversions. Access to 5-thio-d-Fructose and interaction with the d-Fructose transporter, GLUT5
Carbohydrate Research, 2001Co-Authors: Arnaud Tatibouet, Myriam Lefoix, Jonathan Nadolny, Olivier R Martin, Patrick Rollin, Jing Yang, Geoffrey D HolmanAbstract:Abstract Epimerisation and subsequent functionalization at C-5 of d -fructopyranose derivatives under Mitsunobu and Garegg's conditions provided efficient access to 5-thio- d -Fructose ( 2 ) as well as to 5-azido-5-deoxy-1,2- O -isopropylidene-β- d -fructopyranose ( 19 ), a known precursor to 2,5-deoxy-2,5-imino- d -mannitol ( 3 ). The interaction of 2 with the d -Fructose transporter GLUT5, was found to be weaker than that of d -Fructose, a result that suggests involvement of the ring oxygen atom in the recognition of d -Fructose by GLUT5.
Mark H. Rider - One of the best experts on this subject based on the ideXlab platform.
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6 phosphofructo 2 kinase Fructose 2 6 bisphosphatase head to head with a bifunctional enzyme that controls glycolysis
Biochemical Journal, 2004Co-Authors: Mark H. Rider, Guy G Rousseau, Luc Bertrand, Didier Vertommen, Paul A M Michels, Louis HueAbstract:Fru-2,6-P2 (Fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (Fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.
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Signaling pathway involved in the activation of heart 6-phosphofructo-2-kinase by insulin.
Journal of Biological Chemistry, 1996Co-Authors: Véronique Lefebvre, Mark H. Rider, Marie Claire Méchin, Marc P. Louckx, Louis HueAbstract:Incubation of isolated rat cardiomyocytes with insulin increased 2-deoxyglucose uptake, glycogen synthesis, and Fructose 2, 6-bisphosphate content. Half-maximal effects were obtained with 1-2 nM insulin. The insulin-induced increase in Fructose 2,6-bisphosphate content was preceded by a 2-3-fold activation of 6-phosphofructo-2-kinase, which was independent of glucose transport. Insulin activated phosphatidylinositol 3-kinase and p70 ribosomal S6 kinase (p70 S6 kinase), but had no significant effect on mitogen-activated protein kinase, although phorbol 12-myristate 13-acetate activated the latter. The effect of insulin on Fructose 2, 6-bisphosphate, 6-phosphofructo-2-kinase, and phosphatidylinositol 3-kinase was blocked by wortmannin. However, rapamycin, which inhibited p70 S6 kinase activation, and PD 98059, an inhibitor of the mitogen-activated protein kinase pathway, had no effect on the insulin-induced activation of 6-phosphofructo-2-kinase. Heart 6-phosphofructo-2-kinase can therefore be regarded as a glycolytic target of insulin. Its activation by insulin might be mediated by phosphatidylinositol 3-kinase.
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role of Fructose 2 6 bisphosphate in the control of heart glycolysis
Journal of Biological Chemistry, 1993Co-Authors: Christophe Depre, Mark H. Rider, Keith Veitch, Louis HueAbstract:The aim of this work was to study whether changes in Fructose 2,6-bisphosphate concentration are correlated with variations of the glycolytic flux in the isolated working rat heart. Glycolysis was stimulated to different extents by increasing the concentration of glucose, increasing the workload, or by the addition of insulin. The glycolytic flux was measured by the rate of detritiation of [2-3H]- and [3-3H]glucose. Under all the conditions tested, an increase in Fructose 2,6-bisphosphate content was observed. The glucose- or insulin-induced increase in Fructose 2,6-bisphosphate content was related to an increase in the concentration of Fructose 6-phosphate, the substrate of 6-phosphofructo-2-kinase. An increase in the workload correlated with a 50% decrease in the Km of 6-phosphofructo-2-kinase for Fructose 6-phosphate. Similar changes in Km have been observed when purified heart 6-phosphofructo-2-kinase was phosphorylated in vitro by the cyclic AMP-dependent protein kinase or by the calcium/calmodulin-dependent protein kinase. Since the concentration of cyclic AMP was not affected by increasing the workload, it is possible that the change in Km of 6-phosphofructo-2-kinase, which was found in hearts submitted to a high load, resulted from phosphorylation by calcium/calmodulin protein kinase; other possibilities are not excluded. Anoxia decreased the external work developed by the heart, stimulated glycolysis and glycogenolysis, but did not increase Fructose 2,6-bisphosphate.
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inactivation of liver 6 phosphofructo 2 kinase Fructose 2 6 bisphosphatase by phenylglyoxal evidence for essential arginine residues
FEBS Journal, 1992Co-Authors: Mark H. RiderAbstract:Treatment of liver 6-phosphofructo-2-kinase/Fructose-2,6-bisphosphatase with the arginine-specific reagent, phenylglyoxal, irreversibly inactivated both 6-phosphofructo-2-kinase and Fructose-6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against Fructose-2,6-bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic-AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by Fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in Fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme.
