RESUMO
The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10mM for 22h) was added to the maturation medium (P<0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P<0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5mM for 22h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5mM (the highest concentration tested; P<0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P<0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P<0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9h maturation (P<0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos/fisiologia , Via de Pentose Fosfato/fisiologia , 6-Aminonicotinamida/farmacologia , Animais , Bovinos , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Células do Cúmulo/fisiologia , Feminino , Glucose/metabolismo , Ácido Láctico/biossíntese , Meiose/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , NADP/farmacologia , Oócitos/ultraestrutura , Oxirredução , Via de Pentose Fosfato/efeitos dos fármacosRESUMO
Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.
Assuntos
Glicólise/fisiologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Via de Pentose Fosfato/fisiologia , Suínos , 6-Aminonicotinamida/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Células do Cúmulo , Feminino , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Ácido Láctico/biossíntese , NADP/farmacologia , Oócitos/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Fluoreto de Sódio/farmacologiaRESUMO
Cellular energy metabolism is one of the main processes affected during the transition from normal to cancer cells, and it is a crucial determinant of cell proliferation or cell death. As a support for rapid proliferation, cancer cells choose to use glycolysis even in the presence of oxygen (Warburg effect) to fuel macromolecules for the synthesis of nucleotides, fatty acids, and amino acids for the accelerated mitosis, rather than fuel the tricarboxylic acid cycle and oxidative phosphorylation. Mitochondria biogenesis is also reprogrammed in cancer cells, and the destiny of those cells is determined by the balance between energy and macromolecule supplies, and the efficiency of buffering of the cumulative radical oxygen species. In glioblastoma, the most frequent and malignant adult brain tumor, a metabolic shift toward aerobic glycolysis is observed, with regulation by well known genes as integrants of oncogenic pathways such as phosphoinositide 3-kinase/protein kinase, MYC, and hypoxia regulated gene as hypoxia induced factor 1. The expression profile of a set of genes coding for glycolysis and the tricarboxylic acid cycle in glioblastoma cases confirms this metabolic switch. An understanding of how the main metabolic pathways are modified by cancer cells and the interactions between oncogenes and tumor suppressor genes with these pathways may enlighten new strategies in cancer therapy. In the present review, the main metabolic pathways are compared in normal and cancer cells, and key regulations by the main oncogenes and tumor suppressor genes are discussed. Potential therapeutic targets of the cancer energetic metabolism are enumerated, highlighting the astrocytomas, the most common brain cancer.
Assuntos
Neoplasias Encefálicas/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , Oncogenes/fisiologia , Neoplasias Encefálicas/fisiopatologia , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Humanos , Via de Pentose Fosfato/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Cellular energy metabolism is one of the main processes affected during the transition from normal to cancer cells, and it is a crucial determinant of cell proliferation or cell death. As a support for rapid proliferation, cancer cells choose to use glycolysis even in the presence of oxygen (Warburg effect) to fuel macromolecules for the synthesis of nucleotides, fatty acids, and amino acids for the accelerated mitosis, rather than fuel the tricarboxylic acid cycle and oxidative phosphorylation. Mitochondria biogenesis is also reprogrammed in cancer cells, and the destiny of those cells is determined by the balance between energy and macromolecule supplies, and the efficiency of buffering of the cumulative radical oxygen species. In glioblastoma, the most frequent and malignant adult brain tumor, a metabolic shift toward aerobic glycolysis is observed, with regulation by well known genes as integrants of oncogenic pathways such as phosphoinositide 3-kinase/protein kinase, MYC, and hypoxia regulated gene as hypoxia induced factor 1. The expression profile of a set of genes coding for glycolysis and the tricarboxylic acid cycle in glioblastoma cases confirms this metabolic switch. An understanding of how the main metabolic pathways are modified by cancer cells and the interactions between oncogenes and tumor suppressor genes with these pathways may enlighten new strategies in cancer therapy. In the present review, the main metabolic pathways are compared in normal and cancer cells, and key regulations by the main oncogenes and tumor suppressor genes are discussed. Potential therapeutic targets of the cancer energetic metabolism are enumerated, highlighting the astrocytomas, the most common brain cancer.
Assuntos
Humanos , Neoplasias Encefálicas , Glutaminase , Glutamina , Oncogenes/fisiologia , Neoplasias Encefálicas , Proliferação de Células , Transformação Celular Neoplásica , Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Via de Pentose Fosfato/fisiologia , Células-Tronco , Células-TroncoRESUMO
1. Fasting, which increases the catabolism of fatty acids, gives functional protection to the ischaemic-reperfused heart. To obtain further knowledge of this cardioprotective effect, changes in mitochondrial permeability transition (MPT) were measured by the entrapment of 2-deoxy-[(3)H]-glucose (2-DG). We also assessed whether MPT is associated with changes in glutathione status, the activity of glucose-6-phosphate-dehydrogenase (G6PDH) and tissue oxidative damage, estimated by the measurement of Thiobarbituric acid-reactive substances (TBARS). 2. Spontaneously beating hearts of fed and 24 h fasted rats were Langendorff perfused with Krebs'-Ringer bicarbonate solution (10 mmol/L glucose) and exposed to 25 min global ischaemia, followed by 30 min reperfusion. 3. Ischaemia-reperfusion resulted in a fourfold increase in mitochondrial entrapment of 2-DG in the fed group. This response was 29% lower in the fasted group, but there were no concomitant changes in total retention of 2-DG in the heart. Fasting increased the activity of G6PDH by a factor of 1.4 and caused a 2.8-fold increase in the ratio of reduced glutathione to oxidized glutathione (GSH:GSSG) at the end of the pre-ischaemic period. Ischaemia-reperfusion did not affect G6PDH activity, but reduced the GSH:GSSG ratio in both the fed and fasted groups by 50%. Therefore, the GSH:GSSG ratio remained higher in the fasted group. Fasting also decreased cellular levels of TBARS by approximately 25%. Lipolysis of endogenous triacylglycerol was increased during the pre-ischaemic period in the fasted group. 4. These data suggest that the enhancement of fatty acid catabolism that occurs in fasting activates mechanisms that tend to reduce oxidative damage and limit MPT.
Assuntos
Jejum/metabolismo , Glutationa/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Estresse Oxidativo/fisiologia , Via de Pentose Fosfato/fisiologia , Animais , Feminino , Potencial da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Permeabilidade , Ratos , Ratos WistarRESUMO
We investigated the effect of copper on liver key enzymes of the anaerobic glucose metabolism (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK; lactate dehydrogenase, LDH) as well as of the pentose pathway (glycose-6-phosphate dehydrogenase, G6PDH) from the fish Prochilodus lineatus. The fish were acclimated at either 20 degrees C or 30 degrees C at pH 7.0, transferred to water at pH 4.5 or 8.0, and exposed to 96 h-CL(50) copper concentrations. Copper accumulation in liver was higher in fish acclimated at 20 degrees C and maintained in water pH 8.0. Three-way analysis of variance revealed a significant effect of temperature on all enzymes, a significant effect of pH on all enzymes except for PK, and a significant effect of copper on only PFK, and LDH in pH 4.5 at 20 degrees C and, at 30 degrees C, on PFK and PK at pH 4.5 and 8.0, HK at pH 4.5 and G6PDH at pH 8.0. There were significant interactions between treatments for many enzymes. These changes suggest that the activity of enzymes in question is modified by a change in ambient water. At least at 30 degrees C, the overall reduction in the glycolytic enzyme activities of copper-exposed fish seems to reduce energy availability via glucose metabolism, thereby contributing to enhance copper toxic effects.
Assuntos
Anaerobiose/efeitos dos fármacos , Cobre/toxicidade , Peixes/metabolismo , Glucose/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Anaerobiose/fisiologia , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Água Doce , Glucosefosfato Desidrogenase/metabolismo , Hexoquinase/metabolismo , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/fisiologia , Fosfofrutoquinases/metabolismo , Piruvato Quinase/metabolismo , TemperaturaRESUMO
Expression of plasmid-encoded genes in bacteria is the most common strategy for the production of specific proteins in biotechnological processes. However, the synthesis of plasmid-encoded proteins and plasmid-DNA replication often places a metabolic load (metabolic burden) into the cell's biochemical capacities that usually reduces the growth rate of the producing culture (Glick BR. Biotechnol Adv 1995;13:247-261). This metabolic burden may be related to a limited capacity of the cell to supply the extra demand of building blocks and energy required to replicate plasmid DNA and express foreign multicopy genes. Some of these required blocks are intermediaries of the pentose phosphate (PP) pathway, e.g., ribose-5-phosphate, erythrose-4-phosphate. Due to the important impact of metabolic burden on biotechnological processes, several groups have worked on developing strategies to overcome this problem, like reduction of plasmid copy number (Seo JH, Bailey JE. Biotechnol Bioeng 1985;27:1668-1674; Jones KL, Kim S, Keasling JD. Metab Eng 2000;3:328-338), chromosomal insertion of the gene which product is desired, or changing the plasmid-coded antibiotic resistance gene (Hong Y, Pasternak JJ, Glick BR. Can J Microbiol 1995;41:624-628). However, few efforts have been attempted to overcome the reduction of growth rate due to protein over-expression, by modifying central metabolic pathways (Chou C-H, Bennett GN, San KY. Biotechnol Bioeng 1994;44:952-960). We constructed a high-copy number plasmid carrying the gene for glucose-6-phosphate dehydrogenase, zwf, under the control of an inducible trc promoter (pTRzwf04 plasmid). By transforming a wild-type strain and inducing with IPTG, it was possible to recover growth-rate from 0.46 h(-1) (uninduced) to 0.64 h(-1) (induced). The same transformation in an Escherichia coli zwf(-), allows a growth-rate recovery from 0.43 h(-1) (uninduced) to 0.62 h(-1) (induced). We also studied this effect as part of a laboratory-scale biotechnology process: production of a recombinant insulin peptide by co-transforming E. coli JM101 strain with pTRzwf07, a low-copy-number plasmid that carries the same inducible construction as pTRzwf04, and with the pTEXP-MMRPI vector that carries a TrpLE-proinsulin hybrid gene. In this system, production of TrpLE-proinsulin strongly reduces growth rate; however, overexpression of zwf gene recovers with a growth rate from 0.1 h(-1) in the TrpLE-proinsulin induced strain, to 0.37 h(-1) when both zwf and TrpLE-proinsulin genes were induced. In this paper, we show that the engineering of the pentose phosphate pathway by modulation of the zwf gene expression level partially overcomes the possible bottleneck for the supply of building blocks and reducing power synthesized through the PP pathway, that are required for plasmid replication and plasmid-encoded protein expression.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Glucosefosfato Desidrogenase/metabolismo , Via de Pentose Fosfato/fisiologia , Engenharia de Proteínas/métodos , Transdução de Sinais/fisiologia , Proliferação de Células , Ativação Enzimática , Dosagem de Genes , Glucosefosfato Desidrogenase/genética , Plasmídeos/genéticaRESUMO
The effects of copper and cadmium on metabolism through the pentose phosphate pathway were evaluated in Bufo arenarum toad ovary. The effects of the two metals on dehydrogenases from this pathway were evaluated by three experiments: (1) in samples obtained from control females with addition of the metals to the reaction mixture (in vitro), (2) in samples obtained from control females and after long-term exposure of females to 4 and 100 microg/L of Cu or Cd in the incubation media (in vitro after exposure to the metals in vivo), and (3) 14CO2 production through the pentose phosphate pathway was evaluated after [U-14C]glucose microinjection on ovulated oocytes (in vivo after microinjection of the metals). Results from (1) evidenced inhibition of both enzyme activities but only above 1.5 mM Cu and Cd added to the reaction mixture. In (2) both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities decreased in samples from the ovaries of females exposed in vivo to Cu, in a concentration-dependent manner (up to 90% in females exposed to 100 microg/L Cu: 2.12 +/- 1.57 NADPH micromol/min microg protein x 10(-5) vs 19.97 +/- 8.54 in control females). Cd treatment of the toads only rendered an inhibitory effect on 6-phosphogluconate dehydrogenase activity after exposure to 4 microg/L of the bivalent cation. (3) In vivo 14CO2 evolution significantly decreased in oocytes coinjected with 6.3 x 10(-3) mM Cu (calculated intracellular final concentration of the metal injected) and radioactive glucose. Cu and Cd concentration in samples from exposed females were always under detection limit by particle-induced X-ray emission. The results presented here are in agreement with a role for both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities determination as biomarkers of effect and exposure for Cu but not for Cd toxicity.
Assuntos
Biomarcadores/análise , Bufonidae/fisiologia , Cádmio/toxicidade , Cobre/toxicidade , Glucosefosfato Desidrogenase/farmacologia , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/fisiologia , Fosfogluconato Desidrogenase/farmacologia , Poluentes da Água/toxicidade , Animais , Dióxido de Carbono/análise , Feminino , Glucose/metabolismo , Ovário/fisiologiaRESUMO
In order to investigate the influence of cytoskeletal organization and dynamics on cellular biochemistry, a mathematical model was formulated based on our own experimental evidence. The model couples microtubular protein (MTP) dynamics to the glycolytic pathway and its branches: the Krebs cycle, ethanolic fermentation, and the pentose phosphate (PP) pathway. Results show that the flux through glycolysis coherently and coordinately increases or decreases with increased or decreased levels of polymerized MTP, respectively. The rates of individual enzymatic steps and metabolite concentrations change with the polymeric status of MTP throughout the metabolic network. Negative control is exerted by the PP pathway on the glycolytic flux, and the extent of inhibition depends inversely on the polymerization state of MTP, i.e. a high degree of polymerization relieves the negative control. The stability of the model's steady state dynamics for a wide range of variation of metabolic parameters increased with the degree of polymerized MTP. The findings indicate that the organization of the cytoskeleton bestows coherence and robustness to the coordination of cellular metabolism.
Assuntos
Citoesqueleto/metabolismo , Via de Pentose Fosfato/fisiologia , Estabilidade Enzimática , Ácidos Glicéricos/metabolismo , Glicólise , Microtúbulos/metabolismo , Modelos TeóricosRESUMO
The effect of gonadectomy on lymphocyte proliferation and macrophage function (hydrogen peroxide production and phagocytosis capacity) of male and female rats was examined and the results correlated with the activities of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase and phosphate-dependent glutaminase. Also, the reversion of the changes by the treatment with oestrogen or progesterone or a combination of both was addressed. Taken as a whole, ovariectomy reduced hydrogen peroxide production and phagocytosis capacity by macrophages and also lymphocyte proliferation. Castration of male rats reduced the proliferation of lymphocytes and raised macrophage phagocytosis capacity. The effects on macrophage function were correlated with changes in glucose metabolism, particularly, in the activity of glucose-6-phosphate dehydrogenase.