RESUMEN
Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.
Asunto(s)
Técnicas Biosensibles , Fructosa , Glucólisis , Análisis de la Célula Individual , Fluorescencia , Fructosa/análisis , Fructosadifosfatos/análisis , Humanos , Células Secretoras de Insulina/química , Células Secretoras de Insulina/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Análisis de la Célula Individual/métodosRESUMEN
Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), we transplanted the B. subtilis FBP-binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.
Asunto(s)
Fructosadifosfatos/análisis , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Técnicas Biosensibles , Ingeniería Genética , Glucólisis , Metabolómica , Técnicas Analíticas Microfluídicas , Proteómica , Proteínas Represoras/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In this paper, we report a resonance light scattering (RLS) method for the determination of fructose bisphosphates (FBPs) in water solution using fructose 1,6-bisphosphate (F-1,6-BP) as a model analyte without the procedure of extracting target analyte. The method used a type of modified gold nanoparticles (GNPs) as optical probe. The modified GNPs are uranyl-salophen-cysteamine-GNPs (U-Sal-Cy-GNPs) which are obtained through the acylation reaction of carboxylated salophen with cysteamine-capped GNPs (Cy-GNPs) to form Sal-Cy-GNPs and then the chelation reaction of uranyl with tetradentate ligand salophen in the Sal-Cy-GNPs. A FBP molecule is used easily to connect two U-Sal-Cy-GNPs to cause the aggregation of the GNPs by utilizing the specific affinity of uranyl-salophen complex to phosphate group, resulting in the production of strong RLS signal from the system. The amount of FBPs can be determined through detecting the RLS intensity change of the system. A linear range was found to be 2.5 to 75 nmol/L with a detection limit of 0.91 nmol/L under optimal conditions. The method has been successfully used to determine FBPs in real samples with the recoveries of 96.5-103.5 %.
Asunto(s)
Fructosadifosfatos/orina , Oro/química , Nanopartículas del Metal/química , Compuestos Organometálicos/química , Dispersión Dinámica de Luz , Fructosadifosfatos/análisis , Humanos , Límite de Detección , Nanopartículas del Metal/ultraestructuraRESUMEN
Zr4+-doped polydopamine (Zr@PDA) nanozyme with phosphatase-like activity was synthesized by a one-pot hydrothermal method for the first time. Compared with previous representative phosphatase-mimicking nanozymes (i.e., CeO2 NPs, ZrO2 NPs and UiO-66), Zr@PDA not only exhibited higher dispersion stability in water, but also higher catalytical efficiency. Kcat/Km of Zr@PDA is 35 and 12 times that of UiO-66 and ZrO2 NPs, respectively, which would endow the Zr@PDA-based analytical methods with high sensitivity. As a demonstration, a novel colorimetric method based on Zr@PDA nanozyme was developed for sensitive detection of the drug fructose 1,6-diphosphate. The linear range is 1-15 µM with a detection limit as low as 0.38 µM.
Asunto(s)
Colorimetría , Fructosadifosfatos , Indoles , Polímeros , Circonio , Circonio/química , Indoles/química , Polímeros/química , Fructosadifosfatos/análisis , Fructosadifosfatos/química , Colorimetría/métodos , Límite de Detección , Materiales Biomiméticos/químicaRESUMEN
Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.
Asunto(s)
Glucosa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Saccharomyces cerevisiae/enzimología , Apoptosis , Citoplasma/química , Fermentación , Fructosadifosfatos/análisis , Eliminación de Gen , Glucosa-6-Fosfato/análisis , Glucólisis , Concentración de Iones de Hidrógeno , Redes y Vías Metabólicas , Mutación , Saccharomyces cerevisiae/genéticaRESUMEN
Diabetic states are characterized by a raised serum/islet level of long chain fatty acids and a lowered ED50 for glucose-induced insulin secretion. Prolonged culture (> 6 h) of islets with long chain fatty acids replicates the basal insulin hypersecretion. We examined this effect in rat islets cultured for 24 h with 0.25 mM oleate. Insulin secretion at 2.8 mM glucose was doubled in combination with a 60% lowered islet content of glucose-6-phosphate (G6P). Investigation of the lowered G6P showed: (a) increased glucose usage from 0.5 to 100 mM glucose with identical values measured by [2-3H]glucose and [5-3H]glucose, (c) indicating little glucose- 6-phosphatase activity, (b) unchanged low pentose phosphate shunt activity, (c) 50% increased phosphofructokinase (PFK) Vmax, (d) a normal ATP/ADP ratio, and (e) unchanged fructose 2,6 bisphosphate content. Triacsin C, an inhibitor of fatty acyl-CoA synthetase, prevented the increase in PFK activity and the lowered G6P content. These results suggest that long chain acyl-CoA mediates the rise in PFK activity, which in turn lowers the G6P level. We speculate that the inhibition of hexokinase by G6P is thus attenuated, thereby causing the basal insulin hypersecretion.
Asunto(s)
Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ácido Oléico/farmacología , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Regulación Alostérica , Animales , Coenzima A Ligasas/antagonistas & inhibidores , Diabetes Mellitus/metabolismo , Interacciones Farmacológicas , Fructosadifosfatos/análisis , Glucosa-6-Fosfato/análisis , Secreción de Insulina , Islotes Pancreáticos/citología , Oxidación-Reducción , Vía de Pentosa Fosfato , Fosfofructoquinasa-1/metabolismo , Ratas , Ratas Sprague-Dawley , Triazenos/farmacologíaRESUMEN
The rate, key enzymes, and several metabolites of glycolysis in rat hepatoma (HTC) cells have been compared to those in rat hepatocytes. At 5 to 10 mM glucose, lactate release was greater in HTC cells. This could be explained in part by the absence of key gluconeogenic enzymes, by the substitution of glucokinase by hexokinase, and by an increase in phosphofructokinase 1 and pyruvate kinase activity. In addition, fructose 2,6-bisphosphate, the most potent stimulator of phosphofructokinase 1, was identified in HTC cells and shown to stimulate phosphofructokinase 1 partially purified from these cells. Dexamethasone increased the release of lactate in HTC cells. This glucocorticoid increased the concentration of fructose 2,6-bisphosphate and the Vmax of the enzyme that catalyzes its synthesis, phosphofructokinase 2. The data were consistent with an indirect effect at the gene level, mediated by glucocorticoid receptors. Dexamethasone had no effect on the other rate-limiting glycolytic enzymes. Several agents (adenosine, dibutyryl cyclic adenosine 3':5'-monophosphate, ethanol, antimycin) known to decrease fructose 2,6-bisphosphate in hepatocytes were without effect on this stimulator in HTC cells. DL-Glyceraldehyde inhibited glycolysis in HTC cells and eventually killed them. Although this substance decreased fructose 2,6-bisphosphate inhibition of glycolysis through an action at another level could not be ruled out.
Asunto(s)
Fructosadifosfatos/análisis , Glucocorticoides/farmacología , Glucólisis/efectos de los fármacos , Hexosadifosfatos/análisis , Neoplasias Hepáticas Experimentales/metabolismo , Adenosina/farmacología , Animales , Células Cultivadas , Dexametasona/farmacología , Fructosadifosfatos/farmacología , Gliceraldehído/farmacología , Fosfofructoquinasa-1/análisis , RatasRESUMEN
Phosphofructokinase and pyruvate kinase were overexpressed in the filamentous fungus Aspergillus niger. Moderate overexpression of these glycolytic enzymes in A. niger N400 (3-5-fold the wild-type level), either individually or simultaneously, did not increase citric acid production by the fungus significantly. Thus, phosphofructokinase and pyruvate kinase do not seem to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid. Overexpression of phosphofructokinase and pyruvate kinase did not influence the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing phosphofructokinase, the level of fructose 2,6-bisphosphate, a positive allosteric effector of phosphofructokinase, was reduced almost 2-fold compared to the wild-type strain. Measurements with purified phosphofructokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisphosphate level could decrease the specific activity of phosphofructokinase in the cell significantly. Thus, the fungus seems to adapt to overexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bisphosphate.
Asunto(s)
Aspergillus niger/enzimología , Ácido Cítrico/metabolismo , Fosfofructoquinasa-1/biosíntesis , Piruvato Quinasa/biosíntesis , Aspergillus niger/genética , Clonación Molecular , Fructosadifosfatos/análisis , Datos de Secuencia MolecularRESUMEN
5-Iodotubercidin (Itu) and proglycosyn (Pro) have similar glycogenic properties. To compare their mechanisms of action, we tested them in hepatocytes from fasted rats. We show that both compounds are similar in that they stimulated glycogen synthesis, increased the concentration of synthase a, decreased that of phosphorylase a and lowered the concentration of F-2,6-P2 in the presence of glucose, lactate-pyruvate and amino acids. However, when amino acids were absent, Pro was the better stimulator of glycogenesis than Itu and in combination they elevated glycogen and synthase a concentrations synergistically. Further they differ in that (1) Itu enhanced the levels of cyclic AMP whereas Pro did not; (2) Pro depressed glucose production from gluconeogenic substrates, whereas Itu stimulated this process; (3) the inhibition of F-2,6-P2 formation and glycolysis by Pro became much weaker than that by Itu when glucose concentrations were raised from 10 to 20 mM. Inhibition of glycolysis but not that of glycogen synthesis was partly due to a phosphorylated metabolite of Itu. The present study indicates that despite their similar glycogenic effects, Itu and Pro do not share a common mechanism of action. Further, the inhibition of glycolysis and F-2,6-P2 formation by Itu cannot be explained if it acts solely as a general inhibitor of protein kinases.
Asunto(s)
Glucógeno/biosíntesis , Imidazoles/farmacología , Hígado/metabolismo , Tubercidina/análogos & derivados , Animales , Células Cultivadas , AMP Cíclico/análisis , Relación Dosis-Respuesta a Droga , Fructosadifosfatos/análisis , Glucosa/biosíntesis , Glucólisis/efectos de los fármacos , Masculino , Fosforilación , Ratas , Tubercidina/farmacologíaRESUMEN
Effects of phenylephrine, oxytocin and angiotensin on fructose 2,6-bisphosphate (Fru 2,6-P2) content and glycolytic parameters were studied in incubated thymus lymphocytes. These hormones modified Fru 2,6-P2 content dependent upon the energetic status of the cells. In non-preincubated thymus lymphocytes (with relatively high levels of glycogen and ATP), phenylephrine, oxytocin and angiotensin depressed Fru 2,6-P2 content in a dose-dependent manner. The opposite was found when the cells were preincubated for 2 h without substrates (low levels of ATP and glycogen). Changes in lactate release were less evident, but significant. Phenylephrine did not modify the maximal activities of phosphofructokinase (PFK)-1 or PFK-2. However, both submaximal PFK-1 and PFK-2 activities were inhibited by phenylephrine, and the response to exogenous Fru 2,6-P2 on PFK-1 was also altered. The activities of Fru 1,6-P2 and pyruvate kinase were not modified by phenylephrine or A23187 treatment. Simultaneous presence of Cyclosporin A (CsA), an immunosuppressive drug, antagonizes the alpha-adrenergic effect on Fru 2,6-P2 content. CsA alone did not alter basal levels of ATP, hexose phosphate or Fru 2,6-P2, and its opposing effect to alpha-agonist was dose-dependent. CsA cannot change the positive action of PMA or the negative action of A23187 on Fru 2,6-P2 content. The present data suggest that CsA acts prior to calcium liberation and protein kinase C activation. Different possible molecular models are discussed.
Asunto(s)
Angiotensina II/antagonistas & inhibidores , Ciclosporina/farmacología , Glucosa/metabolismo , Linfocitos/efectos de los fármacos , Oxitocina/antagonistas & inhibidores , Fenilefrina/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Calcimicina/farmacología , Relación Dosis-Respuesta a Droga , Fructosadifosfatos/análisis , Glucólisis/efectos de los fármacos , Lactatos/metabolismo , Ácido Láctico , Linfocitos/metabolismo , Masculino , Ratas , Ratas Wistar , Acetato de Tetradecanoilforbol/farmacología , Timo/citologíaRESUMEN
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.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Miocardio/metabolismo , 3-O-Metilglucosa , Animales , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Fructosadifosfatos/análisis , Glucosa/análisis , Glucosa/metabolismo , Glucosa-6-Fosfato , Glucofosfatos/análisis , Corazón/efectos de los fármacos , Humanos , Insulina/farmacología , Masculino , Metilglucósidos/metabolismo , Miocardio/análisis , Fosfofructoquinasa-1/metabolismo , Ratas , Ratas EndogámicasRESUMEN
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.
Asunto(s)
Clorpropamida/farmacología , Fructosadifosfatos/biosíntesis , Gluconeogénesis/efectos de los fármacos , Hexosadifosfatos/biosíntesis , Hígado/efectos de los fármacos , Animales , AMP Cíclico/análisis , Diabetes Mellitus Tipo 2/metabolismo , Fructosadifosfatos/análisis , Glucosa/metabolismo , Humanos , Lactatos/biosíntesis , Ácido Láctico , Hígado/análisis , Hígado/citología , Hígado/metabolismo , Masculino , Fosfofructoquinasa-2 , Fosfotransferasas/metabolismo , Piruvato Quinasa/metabolismo , Piruvatos/metabolismo , Ácido Pirúvico , Ratas , Ratas EndogámicasRESUMEN
Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.
Asunto(s)
Fructosa-Bifosfato Aldolasa/química , Fructosadifosfatos/análisis , Músculos/enzimología , Borohidruros , Simulación por Computador , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Transaldolasa/análisisRESUMEN
Glycolytic flux in skeletal muscle is controlled by 6-phosphofructokinase but how this is achieved is controversial. Brief exercise (swimming) in frogs caused a dramatic increase in the phosphofructokinase activator, fructose 2,6-bisphosphate, in working muscle. The kinetics of phosphofructokinase suggest that in resting muscle, the enzyme is inhibited by ATP plus citrate and that the increase in fructose 2,6-bisphosphate is part of the mechanism to activate phosphofructokinase when exercise begins. When exercise was sustained, fructose 2,6-bisphosphate in muscle was decreased as was the rate of lactate accumulation. Glycolytic flux and the content of fructose 2,6-bisphosphate appear to be closely correlated in working frog muscle in vivo.
Asunto(s)
Anuros/fisiología , Fructosadifosfatos/metabolismo , Glucólisis/fisiología , Hexosadifosfatos/metabolismo , Actividad Motora/fisiología , Músculos/metabolismo , Animales , Activación Enzimática , Fructosadifosfatos/análisis , Cinética , Lactatos/análisis , Lactatos/metabolismo , Ácido Láctico , Músculos/análisis , Fosfofructoquinasa-1/metabolismo , NataciónRESUMEN
The influence of glibenclamide and meglitinide, or 4-[2-(5-chloro-2-methoxybenzamide)ethyl]-benzoic acid, a compound similar to the nonsulfonylurea moiety of glibenclamide, on glycogen phosphorylase a activity, fructose 2,6-bisphosphate (F-2,6-P2) level, and cytoplasmic free-Ca2+ concentration has been studied in isolated rat hepatocytes. Both glibenclamide and meglitinide caused a transient and dose-dependent activation of glycogen phosphorylase, with half-maximal effects corresponding to 3.7 +/- 1.6 and 9.6 +/- 3.3 mumol/L, respectively. This enzyme activation occurred without significant changes in hepatocyte cyclic adenosine monophosphate (cAMP) levels and was accompanied by an increase in cytoplasmic concentration of free Ca2+. Parallel to these effects, glibenclamide increased the cellular content of F-2,6-P2, with this effect being associated with a reduction in the rate of glucose formation from a mixture of [14C]lactate/pyruvate. Under similar conditions, meglitinide caused a significant reduction of F-2,6-P2 levels and accelerated the gluconeogenic flux. The mechanism by which meglitinide decreases hepatocyte F-2,6-P2 levels seems to be mediated by stimulation of fructose-2,6-bisphosphatase. This comparative study may help to elucidate which among the hepatic effects of glibenclamide are exerted specifically by the sulfonylurea moiety.
Asunto(s)
Benzamidas/farmacología , Fructosadifosfatos/análisis , Gliburida/farmacología , Hipoglucemiantes/farmacología , Hígado/efectos de los fármacos , Fosforilasas/análisis , Nucleótidos de Adenina/metabolismo , Animales , Benzamidas/química , Calcio/análisis , Calcio/metabolismo , Calcio/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fructosadifosfatos/metabolismo , Gliburida/química , Hipoglucemiantes/química , Hígado/citología , Hígado/metabolismo , Masculino , Oxidación-Reducción , Fenilefrina/farmacología , Fosforilasas/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Sugar metabolism and exopolysaccharide (EPS) production was analysed in Lactococcus lactis by in vivo 31P NMR. Transient production of several sugar phosphates, transient depletion of intracellular phosphate, transient production of ATP and UTP, transient acidification of the medium and alkalinisation of the cytoplasm could be observed in a period of 20 min upon energization by the addition of glucose. EPS and non-EPS producing variants showed similar NMR spectra, the exception being two pH-dependent resonances observed in the former. They were already observed before addition of glucose and their response to glucose incubation reflected exposure to the medium. They are presumably phosphorylated poly- or oligosaccharides being loosely adhered to cell walls. By freezing and perchloric acid extraction of the cell material, different types of phosphorylated compounds could be recognised in the NMR spectra such as fructose-1-6-diphosphate, nucleotides (like ADP, ATP, UTP and TDP) and several nucleotide sugars. The ongoing work is focused on identifying the unknown peaks and quantifying the differences between wild-type cells and the EPS producing variant.
Asunto(s)
Fructosadifosfatos/metabolismo , Lactococcus lactis/metabolismo , Espectroscopía de Resonancia Magnética , Polisacáridos Bacterianos/biosíntesis , Adenosina Difosfato/análisis , Adenosina Trifosfato/análisis , Microbiología de Alimentos , Fructosadifosfatos/análisis , Genes Bacterianos/fisiología , Lactococcus lactis/química , Lactococcus lactis/genética , Isótopos de Fósforo , Plásmidos/fisiología , Polisacáridos Bacterianos/análisis , Uridina Difosfato/análisis , Uridina Trifosfato/análisisRESUMEN
The effect of troglitazone, an orally effective thiazolidinedione, on lactate- and glucagon-stimulated gluconeogenesis (in the absence of insulin) was examined in hepatocytes isolated from rats under different nutritional states. Hepatocytes obtained from fed or 20-24 hr fasted male Sprague-Dawley rats were incubated in Krebs-Henseleit Bicarbonate buffer (KHBC) (in presence or absence of 10.0 mM glucose) containing 2.0 mM [U-14C]lactate (0.1-0.25 microCi) with or without 10.0 nM glucagon and troglitazone (30.0 microM) or the appropriate vehicle. Aliquots were removed at specified endpoints and assayed for glucose and fructose 2,6-bisphosphate (F-2,6-P2) concentrations. In 20-24 hour starved hepatocytes, troglitazone produced a 26.1% inhibition of lactate-stimulated gluconeogenesis. This inhibitory effect of troglitazone on hepatic gluconeogenesis was further potentiated by incubation of the cells with glucose in vitro. In hepatocytes obtained from fasted rats (and incubated with 10 mM glucose in vitro) troglitazone reduced lactate-and glucagon-stimulated gluconeogenesis by 53% and 56%, respectively. This reduction in hepatic glucose production was associated with 1.06 and 1.04 fold increase in the hepatocyte F-2,6-P2 content. In isolated hepatocytes from fed animals and incubated with 10 mM glucose in vitro, troglitazone (15 and 30 microM) did not have any effect on either lactate- or glucagon-stimulated gluconeogenesis. However, 30 microM troglitazone significantly enhanced (36%) F-2,6-P2 concentrations during lactate-stimulated gluconeogenesis. These findings demonstrate that troglitazone decreases hepatic glucose production through alterations in the activity of one or more gluconeogenic/glycolytic enzymes, depending upon the nutritional state of the animal and the presence or absence of hormonal modulation. All of the effects of troglitazone in the present study were observed in the absence of insulin, suggesting an "insulinomimetic" effect. However, this does not exclude the possibility that troglitazone may also function as an "insulin sensitizer" in hepatic and certain other tissues.
Asunto(s)
Cromanos/farmacología , Fructosadifosfatos/análisis , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Hígado/efectos de los fármacos , Tiazoles/farmacología , Tiazolidinedionas , Animales , Gluconeogénesis/efectos de los fármacos , Glucólisis/efectos de los fármacos , Técnicas In Vitro , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , TroglitazonaRESUMEN
Mature epididymal boar spermatozoa converted fructose, glycerol and glycerol-3-phosphate to carbon dioxide, but in the presence of 0.5 mM 3-bromopyruvate, these oxidations were inhibited while that of lactate was unaffected. Inhibition of the oxidation of these substrates results in a decrease in the content of ATP and the accumulation of dihydroxyacetone phosphate and fructose-1,6-bisphosphate. Examination of the activities of the enzymes within stage two of the glycolytic pathway showed that glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase were immediately inhibited by 3-bromopyruvate in a competitive manner. We now report the action of 3-bromopyruvate on the metabolic activity of boar spermatozoa. At a concentration of 0.5 mM, this compound selectively inhibits stage two of the glycolytic pathway and becomes yet another specific inhibitor of spermatozoal metabolism.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucólisis/efectos de los fármacos , Piruvatos/farmacología , Espermatozoides/efectos de los fármacos , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Animales , Dióxido de Carbono/análisis , Dióxido de Carbono/metabolismo , Relación Dosis-Respuesta a Droga , Fructosa/análisis , Fructosa/metabolismo , Fructosadifosfatos/análisis , Fructosadifosfatos/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Glicerol/análisis , Glicerol/metabolismo , Glicerofosfatos/análisis , Glicerofosfatos/metabolismo , Masculino , Oxidación-Reducción , Fosfoglicerato Quinasa/antagonistas & inhibidores , Espermatozoides/química , Espermatozoides/metabolismo , PorcinosRESUMEN
The activity of 6-phosphofructo-1-kinase (PFK-1), an important regulatory enzyme of glycolysis, was determined after injection of the sialagogue pilocarpine. The fructose-2,6-bisphosphate content of the glands and 6-phosphofructo-2-kinase (PFK-2) activity were also measured. The increase in PFK-1 activity after pilocarpine treatment was likely to be due to the increase in the content of its potent modulator, fructose-2,6-bisphosphate. This in turn was assumed to be due to the increase in the activity of the active form of PFK-2.
Asunto(s)
Fructosadifosfatos/análisis , Glándula Parótida/química , Glándula Parótida/efectos de los fármacos , Fosfofructoquinasa-1/metabolismo , Fosfotransferasas/metabolismo , Pilocarpina/farmacología , Glándula Submandibular/química , Glándula Submandibular/efectos de los fármacos , Animales , Glucógeno/análisis , Masculino , Glándula Parótida/enzimología , Fosfofructoquinasa-2 , Ratas , Ratas Endogámicas , Glándula Submandibular/enzimología , Factores de TiempoRESUMEN
The glycolysis metabolites glucose 6-phosphate (G6-P), fructose 6-phosphate (F6-P), fructose 1,6-bisphosphate (F1,6-BP), fructose 2,6-bisphosphate (F2,6-BP), glyceraldehyde phosphate (GAP), dihydroxyacetone phosphate (DHAP), phosphoenolpyruvate (PEP), pyruvate, and lactate were analyzed by capillary zone electrophoresis (CZE) with indirect UV detection. The chromophores phthalic acid, sorbic acid, and 4-hydroxybenzoic acid were studied as background electrolytes. Both detection sensitivity and resolution were found to depend on the pH and the concentration of the carrier electrolyte. Optimum separation and detection of the phosphate compounds were accomplished upon reversal of electroendosmotic flow (EOF) with OFM Anion-BT (Waters Corp., Milford, MA) at a concentration of 4-6 mM 4-hydroxybenzoic acid, pH 11.6-12.0, with the detection wave-length set at 280 nm. The highly alkaline pH allowed the successful separation of the isomers F6-P and G6-P, as well as F1,6-BP and F2,6-BP, respectively. The effect of sample ionic strength on the detection limits of G6-P, F6-P, F1,6-BP, and F2,6-BP was also investigated: These limits ranged from 1 to 3 microM in both low- and high-ionic-strength samples. However, high Mg2+ concentrations in the sample led to a progressive loss of resolution between F1,6-BP and F2,6-BP, unless the inlet reservoir was replenished with fresh electrolyte after every injection. Linearity of detection was observed over one to two orders of magnitude.