ABSTRACT
It is known that both interleukin-1 alpha (IL-1 alpha) and 12-O-tetradecanoylphorbol 13-acetate (TPA) promote increases in intracellular levels of the glycolytic regulatory metabolite fructose 2,6-bisphosphate [Fru(2,6)P2] and in the production of prostaglandin E (PGE) by subcultured rheumatoid synovial cells (RSC) and human dermal fibroblasts in vitro. We report here that the protein kinase C inhibitor staurosporine enhanced the IL-1 alpha-induced increase in [Fru(2,6)P2] and PGE production by RSC, whereas in similar concentrations (3-30 nM) this inhibitor decreased the TPA-induced stimulation of these parameters. Staurosporine produced a similar enhancement of the response to IL-1 alpha by normal human dermal fibroblasts. The increased PGE production provoked by tumour necrosis factor alpha (TNF alpha) in RSC was also augmented by staurosporine, but, in contrast, the increases in cellular [Fru(2,6)P2] induced by transforming growth factor beta (TGF beta) and interferon-gamma (IFN-gamma) were diminished. Thus the protein kinase C inhibitor staurosporine discriminates not only between the effects produced by IL-1 alpha and TPA, but also between those of IL-1 alpha and two other cytokines (but not between IL-1 alpha and TNF alpha). These findings suggest that IL-1 alpha and probably TNF alpha act via an intracellular mechanism different from that mediating the action of TPA, TGF-beta and IFN-gamma, and provide evidence that staurosporine is capable of amplifying the IL-1 signal.
Subject(s)
Alkaloids/pharmacology , Biological Factors/pharmacology , Fibroblasts/metabolism , Fructosediphosphates/biosynthesis , Hexosediphosphates/biosynthesis , Prostaglandins E/biosynthesis , Protein Kinase C/antagonists & inhibitors , Cells, Cultured , Cytokines , Fibroblasts/drug effects , Fibroblasts/enzymology , Humans , Interferon-gamma/pharmacology , Interleukin-1/pharmacology , Skin/cytology , Staurosporine , Stimulation, Chemical , Synovial Fluid/cytology , Tetradecanoylphorbol Acetate/pharmacology , Transforming Growth Factors/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The effect of ACTH on glycolysis has been studied in Y-1 tumor adrenal cells. ACTH caused a sustained increase in the liberation of lactate as well as a stimulation of both basal and glucose-induced fructose 2,6-bisphosphate content. ACTH produces changes also in the activities of phosphofructokinase-1 and phosphofructokinase-2. The addition of Ca2+ or dibutyryl cyclic AMP did not modify neither lactate production nor fructose 2,6-bisphosphate levels. The results suggest that fructose 2,6-bisphosphate regulates ACTH-induced glycolysis at the phosphofructokinase-1 step, although the biochemical mechanism of phosphofructokinase-2 activation remains elusive.
Subject(s)
Adrenal Gland Neoplasms/metabolism , Adrenocorticotropic Hormone/pharmacology , Fructosediphosphates/biosynthesis , Glycolysis/drug effects , Hexosediphosphates/biosynthesis , Adrenal Gland Neoplasms/enzymology , Animals , Bucladesine/pharmacology , Calcium/pharmacology , Fructosediphosphates/physiology , Glucose/pharmacology , Lactates/metabolism , Phosphofructokinase-1/metabolism , Tumor Cells, CulturedABSTRACT
The addition of either recombinant human interleukin 1 (IL1 alpha) or 12-O-tetradecanoyl phorbol-13-acetate (TPA) to cultured rheumatoid synovial cells (RSC) caused dose-related increases in PGE production and cellular fructose 2,6-bisphosphate (Fru-2,6-P2). IL1 consistently produced the greater increases in both parameters. A close association between increases in PGE production and Fru-2,6-P2 was demonstrated for both IL1- and TPA-stimulated cells. The combined addition of IL1 with TPA resulted in an additive increase in both parameters. When IL1 was added together with human recombinant interferon-gamma (IFN-gamma), the resulting Fru-2,6-P2 level was synergistically increased, whilst the combination of IFN-gamma and TPA produced only an additive increase. Thus despite their very similar effects on RSC in culture, the data suggests that IL1 and TPA do not act via an identical intracellular mechanism.
Subject(s)
Fructosediphosphates/biosynthesis , Hexosediphosphates/biosynthesis , Interleukin-1/pharmacology , Prostaglandins E/biosynthesis , Rheumatic Diseases/metabolism , Synovial Membrane/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Cells, Cultured , Drug Synergism , Humans , Interferon-gamma/pharmacology , Recombinant Proteins/pharmacology , Synovial Membrane/drug effectsABSTRACT
The effects of tolbutamide and pancreatic hormones on liver fructose-2,6-bisphosphate (F-2,6-P2) formation were examined using isolated rat hepatocytes. Glucagon decreased the F-2,6-P2 level in a dose-dependent manner. Insulin (greater than 10(-9) M) increased the F-2,6-P2 level reduced by glucagon (less than 10(-9) M), but did not show a stimulatory effect on this activator formation in the absence of glucagon. On the other hand, tolbutamide increased the F-2,6-P2 level in hepatocytes regardless of the presence or absence of glucagon. Tolbutamide (2 mM) stimulation on liver F-2,6-P2 formation was enhanced by the concomitant addition of insulin (10(-8) M) in the presence of glucagon (3 X 10(-11) M). These observations suggest that the regulatory effect of tolbutamide on liver F-2,6-P2 level is independent of that of insulin.
Subject(s)
Fructosediphosphates/biosynthesis , Hexosediphosphates/biosynthesis , Insulin/pharmacology , Liver/metabolism , Tolbutamide/pharmacology , Animals , Glucagon/pharmacology , In Vitro Techniques , Kinetics , Liver/drug effects , Male , Rats , Rats, Inbred StrainsABSTRACT
The effects of dichloroacetate and phenazine methosulphate on the content of fructose-2,6-bisphosphate and glycogenesis in incubated epididymal adipose tissue were examined. Both agents stimulated the synthesis of fructose-2,6-bisphosphate in the presence of glucose, the effect being higher in tissue from fasted-refed rats than in normal fed rats. Additions of dichloroacetate to the incubation medium also increased the incorporation of [U-14C]glucose into glycogen and this effect was additive with that of insulin. However phenazine methosulphate strongly depressed the insulin-dependent glycogen synthesis. These data are considered in relation to the increased rate of glucose metabolism known to occur in the presence of dichloroacetate and the stimulation of pentose phosphate pathway with phenazine methosulphate.
Subject(s)
Acetates/pharmacology , Adipose Tissue/metabolism , Dichloroacetic Acid/pharmacology , Fructosediphosphates/biosynthesis , Glycogen/biosynthesis , Hexosediphosphates/biosynthesis , Methylphenazonium Methosulfate/pharmacology , Phenazines/pharmacology , Adipose Tissue/drug effects , Animals , Epididymis , Insulin/pharmacology , Kinetics , Male , Rats , Rats, Inbred StrainsABSTRACT
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.
Subject(s)
Chlorpropamide/pharmacology , Fructosediphosphates/biosynthesis , Gluconeogenesis/drug effects , Hexosediphosphates/biosynthesis , Liver/drug effects , Animals , Cyclic AMP/analysis , Diabetes Mellitus, Type 2/metabolism , Fructosediphosphates/analysis , Glucose/metabolism , Humans , Lactates/biosynthesis , Lactic Acid , Liver/analysis , Liver/cytology , Liver/metabolism , Male , Phosphofructokinase-2 , Phosphotransferases/metabolism , Pyruvate Kinase/metabolism , Pyruvates/metabolism , Pyruvic Acid , Rats , Rats, Inbred StrainsABSTRACT
Incubation of chicken embryo fibroblasts with mitogenic concentrations of insulin for 24 hr or with the tumor promoter phorbol 12-myristate 13-acetate for 6 hr stimulated lactate release and 3-O-methylglucose uptake. Insulin also increased the Vmax of 6-phosphofructo-1-kinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11). Both agents increased the concentration of fructose 2,6-bisphosphate and the activity of 6-phosphofructo-2-kinase (EC 2.7.1.-), the enzyme that catalyzes the synthesis of this stimulator of 6-phosphofructo-1-kinase. These changes provide an explanation for the stimulation of glycolysis by insulin and phorbol esters. In contrast to the situation in rat liver, fructose 2,6-bisphosphate concentration did not decrease after cyclic AMP treatment. Incubation of cells with phorbol ester analogues or with glycerol derivatives that are known to stimulate, or to bind to, protein kinase C did increase the concentration of fructose 2,6-bisphosphate, suggesting that the stimulation of 6-phosphofructo-2-kinase by phorbol 12-myristate 13-acetate is mediated by protein kinase C.
Subject(s)
Carcinogens/pharmacology , Fructosediphosphates/biosynthesis , Glycolysis/drug effects , Hexosediphosphates/biosynthesis , Insulin/pharmacology , Phorbols/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , 3-O-Methylglucose , Animals , Cells, Cultured , Chick Embryo , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Fibroblasts/metabolism , Glycerol/analogs & derivatives , Glycerol/pharmacology , Lactates/analysis , Methylglucosides/metabolism , Phosphofructokinase-1/metabolismABSTRACT
The effects of 5 sulfonylureas on fructose-2,6-bisphosphate (F-2,6-P2) formation using isolated perfused rat liver were examined. All sulfonylureas examined stimulated dose-dependent formation of the activator in a limited range of the concentration. A maximum effect on F-2,6-P2 formation was observed at the concentration of 10(-3) M of tolbutamide or chlorpropamide, at 10(-4) M of gliclazide or acetohexamide and at 10(-6) M of glibenclamide. These concentrations of sulfonylurea correspond with those in blood when therapeutical doses of the drug are administered orally. Sulfonamide and biguanide did not show any stimulatory effect on F-2,6-P2 level. The results demonstrate that stimulation of liver F-2,6-P2 formation is a common characteristic of sulfonylureas and suggest strongly that one of the extrapancreatic actions of sulfonylurea is stimulation of F-2,6-P2 formation followed by enhancement of glycolysis and inhibition of gluconeogenesis in the liver.
Subject(s)
Fructosediphosphates/biosynthesis , Hexosediphosphates/biosynthesis , Liver/metabolism , Sulfonylurea Compounds/pharmacology , Acetohexamide/pharmacology , Animals , Buformin/pharmacology , Chlorpropamide/pharmacology , Gliclazide/pharmacology , Gluconeogenesis/drug effects , Glyburide/pharmacology , Glycolysis/drug effects , In Vitro Techniques , Liver/drug effects , Male , Rats , Rats, Inbred Strains , Stimulation, Chemical , Sulfamethoxazole/pharmacology , Tolbutamide/pharmacologyABSTRACT
The participation of fructose 2,6-bisphosphate on glycolysis stimulated by insulin and adrenaline in incubated white adipose tissue of rat was investigated. Adrenaline addition to incubated fat-pads strongly decreased the intracellular levels of fructose 2,6-bisphosphate. When the tissue was preincubated with glucose, the presence of insulin in the incubation medium increased fructose 2,6-bisphosphate levels 2-fold. These variations were related to changes in the substrates, ATP and fructose 6-phosphate. It therefore appears that fructose 2,6-bisphosphate may be involved in the control of insulin-induced glycolysis, but it does not seem to play a role in the stimulation of glucolysis by adrenaline.
Subject(s)
Adipose Tissue/metabolism , Epinephrine/pharmacology , Fructosediphosphates/biosynthesis , Hexosediphosphates/biosynthesis , Insulin/pharmacology , Adenosine Triphosphate/metabolism , Adipose Tissue/drug effects , Animals , Fructosephosphates/metabolism , Glucose/pharmacology , Glycolysis , Kinetics , Male , Rats , Rats, Inbred StrainsABSTRACT
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.
Subject(s)
Fructosediphosphates/biosynthesis , Hexosediphosphates/biosynthesis , Liver/metabolism , Tolbutamide/pharmacology , Animals , Dose-Response Relationship, Drug , Glucagon/metabolism , Glucose/pharmacology , In Vitro Techniques , Kinetics , Liver/drug effects , Male , Perfusion , Rats , Rats, Inbred StrainsSubject(s)
Fructosediphosphates/biosynthesis , Hexosediphosphates/biosynthesis , Liver/metabolism , Phosphofructokinase-1/isolation & purification , Adenosine Triphosphate , Animals , Cytosol/metabolism , Fructosephosphates , Magnesium , Male , Molecular Weight , Rats , Rats, Inbred Strains , Substrate SpecificitySubject(s)
Adenosine Triphosphate/metabolism , Fructosediphosphates/biosynthesis , Fructosephosphates/metabolism , Hexosediphosphates/biosynthesis , Phosphofructokinase-1/metabolism , Animals , Citrates/pharmacology , Citric Acid , Kinetics , Liver/metabolism , Phosphates/pharmacology , Phosphofructokinase-1/antagonists & inhibitors , Phosphofructokinase-1/isolation & purification , RatsABSTRACT
We have recently discovered an activator for phosphofructokinase termed "activation factor" (Furuya, E., and Uyeda, K. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5861-5864). In this paper, we investigated the enzyme responsible for its synthesis. We have found an enzyme from rat liver which catalyzes the formation of activation factor from fructose-6-P and ATP-Mg and it has been identified as fructose-2,6-P2. Fructose-1,6-P2, fructose-1-P, or fructose does not serve as a substrate. This enzyme has been partially purified and shown to be different from phosphofructokinase. Several lines of evidence indicate that the in vitro synthetic product is identical with chemically synthesized fructose-2,6-P2: (a) it is active in our assay for activation factor which is based on counteraction of ATP inhibition of phosphofructokinase; (b) it is acid labile as is fructose-2,6-P2; and (c) it shows the same mobility as synthetic fructose-2,6-P2 upon paper chromatography and the acid hydrolysis product has been identified as fructose-6-P. Thus, this new enzyme catalyzes the synthesis of the activation factor from fructose-6-P and ATP-Mg.
Subject(s)
Adenosine Triphosphate/metabolism , Fructosediphosphates/biosynthesis , Fructosephosphates/metabolism , Hexosediphosphates/biosynthesis , Liver/metabolism , Phosphofructokinase-1/metabolism , Animals , Enzyme Activation , Kinetics , RatsSubject(s)
Hexosediphosphates/biosynthesis , Phosphotransferases , Acetylglucosamine , Glucosephosphates , Kinetics , MethodsABSTRACT
The reaction: glycerate-1,3-P2 PLUS GLUCOSE-1-P YIELDS TO GLUCOSE-1,6-P2 plus glycerate-P is catalyzed by a distinct enzyme of mouse brain. A divalent metal requirement was shown when the enzyme was treated with imidazole and EDTA. Mg2+, Mn2+, Ca2+, Zn2+, Ni2+, Co2+, and Cd2+ were quite effective cofactors. The enzyme, in better than 50 percent yield, has been purified away from 99 percent of the phosphoglucomutase, phosphoglycrate mutase, and phosphofructokinase. Acetyl-P, ATP, enolpyruvate-P, creatine-P, and fructose-1,6-P2 are not phosphoryl donors. Glucose-6-P and mannose-1-P are good alternate acceptors. Mannose-6-P, galactose-Ps, and fructose-Ps have little or no acceptor activity. Strong inhibition was found with fructose-1,6-P2, glycerate-2,3-P2, enolpyruvate-P, and acetyl CoA. From the amount of activity and the kinetic constants of the purified enzyme it seems likely that this enzyme is responsible for the glucose-1,6-P2 synthesis of brain.
Subject(s)
Brain/enzymology , Glucosephosphates/biosynthesis , Hexosediphosphates/biosynthesis , Phosphotransferases/metabolism , Acetyl Coenzyme A/pharmacology , Animals , Cations, Divalent/pharmacology , Diphosphoglyceric Acids/metabolism , Fructosephosphates/pharmacology , Hydrogen-Ion Concentration , Kinetics , Mannose/metabolism , Mice , Phosphoenolpyruvate/pharmacologySubject(s)
Fructosephosphates/analysis , Ketoses/analysis , Animals , Chromatography, Ion Exchange , Chromatography, Thin Layer , Colorimetry , Fructosephosphates/biosynthesis , Fructosephosphates/isolation & purification , Hexokinase , Hexosediphosphates/biosynthesis , Ketoses/isolation & purification , Ketoses/metabolism , Methods , Muscles/enzymology , Phosphofructokinase-1 , Pseudomonadaceae/analysis , Rabbits , Saccharomyces cerevisiae/enzymologySubject(s)
Fructosephosphates/biosynthesis , Hexosediphosphates/biosynthesis , Alkaline Phosphatase , Animals , Chromatography, Ion Exchange , Chromatography, Thin Layer , Escherichia coli/enzymology , Fructose-Bisphosphatase , Fructose-Bisphosphate Aldolase/metabolism , Fructosephosphates/metabolism , Glucose-6-Phosphate Isomerase/metabolism , Glucosephosphate Dehydrogenase , Glycerolphosphate Dehydrogenase , Hexosediphosphates/metabolism , Ketoses/metabolism , Kinetics , Muscles/enzymology , Phosphofructokinase-1/metabolism , Phosphogluconate Dehydrogenase/metabolism , Pyruvate Kinase , Saccharomyces cerevisiae/enzymology , Spectrophotometry, Ultraviolet , Time FactorsSubject(s)
Hexokinase , Hexoses/chemical synthesis , Saccharomyces cerevisiae/enzymology , Acetals , Acetobacter , Acid Phosphatase , Adenosine Triphosphate , Aluminum , Animals , Binding Sites , Chemical Phenomena , Chemistry , Chromatography, Paper , Ethers, Cyclic , Hexosediphosphates/biosynthesis , Hexosephosphates/biosynthesis , Ketones , Kinetics , Lithium , Muscles/enzymology , Phosphofructokinase-1 , Plants/enzymology , Rabbits , Structure-Activity Relationship , Sugar AlcoholsABSTRACT
Previous studies showed that Arthrobacter pyridinolis can transport and utilize d-glucose only after prior growth on certain Krebs cycle intermediates. In contrast, we found that d-fructose was taken up and metabolized by A. pyridinolis without special prior conditions of growth. d-Fructose was first converted to d-fructose-1-phosphate by a phosphoenolpyruvate (PEP):D-fructose phosphotransferase. This activity required both supernatant and pellet fractions from d-fructose-grown cells centrifuged at 150,000 x g. The d-fructose-1-phosphate formed was converted to d-fructose-1, 6-diphosphate. Mutants deficient in PEP:d-fructose phosphotransferase and d-fructose-1-phosphate kinase, or d-fructose-1, 6-diphosphatase (FDPase) were unable to grow on d-fructose but retained the normal ability to use d-glucose. Mutants forming reduced amounts of FDPase were completely unable to grow on d-fructose but were still capable of limited growth on Krebs cycle intermediates. A requirement for higher levels of FDPase for growth on d-fructose than for growth on Krebs cycle intermediates was also indicated by the higher specific activities of FDPase in d-fructose-grown cells than in cells grown on l-malate or amino acids.