RESUMO
To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes.
Assuntos
Proteínas Arqueais/metabolismo , Frutose-Bifosfatase/metabolismo , Methanosarcina/metabolismo , Fosfofrutoquinase-1/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Galinhas , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/isolamento & purificação , Frutosefosfatos/metabolismo , Genes Arqueais , Cinética , Methanosarcina/genética , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/isolamento & purificação , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
The aim of this work was to investigate the effects of chronic treatment with the combination of ezetimibe and simvastatin on gluconeogenesis in rat liver. Rats were treated daily for 28 days with the combination of ezetimibe and simvastatin (10/40 mg/kg) by oral gavage. To measure gluconeogenesis and the associated pathways, isolated perfused rat liver was used. In addition, subcellular fractions, such as microsomes and mitochondria, were used for complementary measures of enzymatic activities. Treatment with the combination of simvastatin and ezetimibe resulted in a decrease in gluconeogenesis from pyruvate (-62%). Basal oxygen consumption of the treated animals was higher (+22%) than that of the control rats, but the resulting oxygen consumption that occurred after pyruvate infusion was 43% lower in animals treated with the combination of simvastatin and ezetimibe. Oxygen consumption in the livers from treated animals was completely inhibited by cyanide (electron transport chain inhibitor), but not by proadifen (cytochrome P450 inhibitor). Chronic treatment with ezetimibe/simvastatin decreased the activity of the key enzymes glucose-6-phosphatase and fructose-1,6-bisphosphatase by 59% and 45%, respectively, which is probably the major reason for the decreased gluconeogenesis seen in ezetimibe-/simvastatin-treated rats. It is also possible that part of the effect of this combination on gluconeogenesis and on the oxygen consumption is related to the impairment of mitochondrial energy transduction.
Assuntos
Ezetimiba/farmacologia , Gluconeogênese/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Cianetos/farmacologia , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Frutose-Bifosfatase/metabolismo , Glucose-6-Fosfatase/metabolismo , Fígado/citologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Proadifeno/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes.
Assuntos
Metabolismo Energético , Frutose-Bifosfatase/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Gluconeogênese , Glicólise , Hepatócitos/metabolismo , Modelos Biológicos , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Frutose-Bifosfatase/química , Frutose-Bifosfatase/genética , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/genética , Células HeLa , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Microscopia Confocal , Transporte Proteico , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismoRESUMO
This study investigated the effects of prolonged exposure of silver catfish (Rhamdia quelen) to the essential oil (EO) of Hesperozygis ringens. Ventilatory rate (VR), stress and metabolic indicators, energy enzyme activities, and mRNA expression of adenohypophyseal hormones were examined in specimens that were exposed for 6 h to 0 (control), 30 or 50 µL L(-1) EO of H. ringens in water. Reduction in VR was observed in response to each treatment, but no differences were found between treatments. Plasma glucose, protein, and osmolality increased in fish exposed to 50 µL L(-1). Moreover, lactate levels increased after exposure to both EO concentrations. Plasma cortisol levels were not changed by EO exposure. Fish exposed to 30 µL L(-1) EO exhibited higher glycerol-3-phosphate dehydrogenase (G3PDH) activity, while exposure to 50 µL L(-1) EO elicited an increase in glucose-6-phosphate dehydrogenase (G6PDH), fructose-biphosphatase (FBP), and 3-hydroxyacyl-CoA-dehydrogenase (HOAD) activities compared with the control group. Expression of growth hormone (GH) only decreased in fish exposed to 50 µL L(-1) EO, while somatolactin (SL) expression decreased in fish exposed to both concentrations of EO. Exposure to EO did not change prolactin expression. The results indicate that GH and SL are associated with energy reorganization in silver catfish. Fish were only slightly affected by 30 µL L(-1) EO of H. ringens, suggesting that it could be used in practices where a reduction in the movement of fish for prolonged periods is beneficial, i.e., such as during fish transportation.
Assuntos
Aquicultura/métodos , Peixes-Gato/fisiologia , Lamiaceae/química , Óleos Voláteis/efeitos adversos , Estresse Fisiológico/efeitos dos fármacos , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Análise de Variância , Animais , Glicemia/efeitos dos fármacos , Proteínas Sanguíneas/efeitos dos fármacos , Primers do DNA/genética , Proteínas de Peixes/metabolismo , Frutose-Bifosfatase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Glicoproteínas/metabolismo , Hormônio do Crescimento/metabolismo , Hidrocortisona/metabolismo , Concentração Osmolar , Consumo de Oxigênio/efeitos dos fármacos , Hormônios Hipofisários/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Espectrofotometria/veterinária , Estresse Fisiológico/fisiologiaRESUMO
BACKGROUND: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure. METHODS: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. RESULTS: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. CONCLUSIONS: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. GENERAL SIGNIFICANCE: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.
Assuntos
Biocatálise , Frutose-Bifosfatase/química , Rim/enzimologia , Animais , Sequência de Bases , Sítios de Ligação , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/metabolismo , Frutosedifosfatos/química , Dados de Sequência Molecular , Subunidades Proteicas , Especificidade por Substrato , SuínosRESUMO
Using a streptozotocin-induced type 1 diabetic rat model, we analyzed and separated the effects of hyperglycemia and hyperinsulinemia over the in vivo expression and subcellular localization of hepatic fructose 1,6-bisphosphatase (FBPase) in the multicellular context of the liver. Our data showed that FBPase subcellular localization was modulated by the nutritional state in normal but not in diabetic rats. By contrast, the liver zonation was not affected in any condition. In healthy starved rats, FBPase was localized in the cytoplasm of hepatocytes, whereas in healthy re-fed rats it was concentrated in the nucleus and the cell periphery. Interestingly, despite the hyperglycemia, FBPase was unable to accumulate in the nucleus in hepatocytes from streptozotocin-induced diabetic rats, suggesting that insulin is a critical in vivo modulator. This idea was confirmed by exogenous insulin supplementation to diabetic rats, where insulin was able to induce the rapid accumulation of FBPase within the hepatocyte nucleus. Besides, hepatic FBPase was found phosphorylated only in the cytoplasm, suggesting that the phosphorylation state is involved in the nuclear translocation. In conclusion, insulin and not hyperglycemia plays a crucial role in the nuclear accumulation of FBPase in vivo and may be an important regulatory mechanism that could account for the increased endogenous glucose production of liver of diabetic rodents.
Assuntos
Núcleo Celular/enzimologia , Diabetes Mellitus Experimental/enzimologia , Frutose-Bifosfatase/metabolismo , Fígado/enzimologia , Animais , Frutose-Bifosfatase/análise , Insulina/farmacologia , Fígado/efeitos dos fármacos , Masculino , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 µM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.
Assuntos
Ácidos Graxos/farmacologia , Fibroblastos/metabolismo , Proteoma/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Ácidos Graxos/fisiologia , Fibroblastos/efeitos dos fármacos , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Expressão Gênica , Camundongos , Células NIH 3T3 , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Proteoma/genética , Proteômica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The causative agent of Chagas disease, Trypanosoma cruzi, metabolizes glucose through two major pathways: glycolysis and the pentose phosphate pathway. Glucose is taken up via one facilitated transporter and its catabolism by the glycolytic pathway leads to the excretion of reduced products, succinate and l-alanine, even in the presence of oxygen; the first six enzymes are located in a peroxisome-like organelle, the glycosome, and the lack of regulatory controls in hexokinase and phosphofructokinase results in the lack of the Pasteur effect. All of the enzymes of the pentose phosphate pathway are present in the four major stages of the parasite's life cycle, and some of them are possible targets for chemotherapy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase are present, but there is no reserve polysaccharide.
Assuntos
Enzimas/metabolismo , Glucose/metabolismo , Trypanosoma cruzi/metabolismo , Alanina/metabolismo , Animais , Doença de Chagas/parasitologia , Frutose-Bifosfatase/metabolismo , Humanos , Microcorpos/metabolismo , Via de Pentose Fosfato , Trypanosoma cruzi/patogenicidadeRESUMO
The actions of tamoxifen, a selective estrogen receptor modulator used in chemotherapy and chemo-prevention of breast cancer, on glycolysis and gluconeogenesis were investigated in the isolated perfused rat liver. Tamoxifen inhibited gluconeogenesis from both lactate and fructose at very low concentrations (e.g., 5µM). The opposite, i.e., stimulation, was found for glycolysis from both endogenous glycogen and fructose. Oxygen uptake was unaffected, inhibited or stimulated, depending on the conditions. Stimulation occurred in both microsomes and mitochondria. Tamoxifen did not affect the most important key-enzymes of gluconeogenesis, namely, phosphoenolpyruvate carboxykinase, pyruvate carboxylase, fructose 1,6-bisphosphatase and glucose 6-phosphatase. Confirming previous observations, however, tamoxifen inhibited very strongly NADH- and succinate-oxidase of freeze-thawing disrupted mitochondria. Tamoxifen promoted the release of both lactate dehydrogenase (mainly cytosolic) and fumarase (mainly mitochondrial) into the perfusate. Tamoxifen (200µM) clearly diminished the ATP content and increased the ADP content of livers in the presence of lactate with a diminution of the ATP/ADP ratio from 1.67 to 0.79. The main causes for gluconeogenesis inhibition are probably: (a) inhibition of energy metabolism; (b) deviation of intermediates (malate and glucose 6-phosphate) for the production of NADPH required in hydroxylation and demethylation reactions; (c) deviation of glucosyl units toward glucuronidation reactions; (d) secondary inhibitory action of nitric oxide, whose production is stimulated by tamoxifen; (e) impairment of the cellular structure, especially the membrane structure. Stimulation of glycolysis is probably a compensatory phenomenon for the diminished mitochondrial ATP production. The multiple actions of tamoxifen at relatively low concentrations can represent a continuous burden to the overall hepatic functions during long treatment periods.
Assuntos
Gluconeogênese/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Fígado/efeitos dos fármacos , Tamoxifeno/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Metabolismo Energético , Frutose/metabolismo , Frutose-Bifosfatase/metabolismo , Fumarato Hidratase/metabolismo , Glucose/metabolismo , Glucose-6-Fosfatase/metabolismo , Glicogênio/metabolismo , Lactato Desidrogenases/metabolismo , Ácido Láctico/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , NAD/metabolismo , Óxido Nítrico/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Carboxilase/metabolismo , Ratos , Ratos WistarRESUMO
The present study assesses the effects of osmotic stress on phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) activities and (14)C-total lipid synthesis from (14)C-glycine in the anterior and posterior gills, jaw muscle, and hepatopancreas of Neohelice granulata. In posterior gills, 24-h exposure to hyperosmotic stress increased PEPCK, FBPase and G6Pase activities. Increase in (14)C-lipid synthesis was associated to the decrease in PEPCK activity after 72-h exposure to hyperosmotic stress. Hypo-osmotic stress decreased PEPCK and G6Pase activities in posterior gills; however, (14)C-lipids increased after 72-h exposure to stress. In anterior gills, decreases in the G6Pase activity after 72-h of hyperosmotic stress and in (14)C-lipogenesis after 144-h were observed, while PEPCK activity increased after 144 h. Exposure to hypo-osmotic stress increased (14)C-lipid synthesis and PEPCK activity in anterior gills. Muscle G6Pase activity increased after 72-h exposure to hypo-osmotic stress; however, no significant change was observed in the lipogenesis. PEPCK decreased in muscle after 144-h exposure to hyperosmotic, coinciding with increased (14)C-lipid synthesis. In the hepatopancreas, a decrease in the (14)C-lipogenesis occurred after 24-h exposure to hyperosmotic stress, accompanied by increase in (14)C-lipid synthesis. Additionally, PEPCK activity returned to control levels. The hepatopancreatic lipogenesis from amino acids was not involved in the metabolic adjustment during hypo-osmotic stress. However, gluconeogenesis is one of the pathways involved in the adjustment of the intracellular concentration of nitrogenated compounds.