Regulation of key enzymes of glucose metabolism in bovine COCs

The aim of this work was to study the regulation of PFK 1 and G6PDH, two key enzymes involved in glucose metabolism in cumulus oocyte-complexes (COCs), and its relationship with the oocyte maturation process. It was observed that the activity of PFK 1 in the presence of ATP was inhibited whereas the addition of AMP increased the activity (P < 0.05). To verify the effect of the physiological modulators on the COC glycolytic pathway, the lactate production during IVM and the maturation rate were evaluated. In accordance with the enzymatic activity, the glycolytic activity evaluated by lactate production and the maturation rate diminished (P < 0.05) with the addition of ATP. While the AMP had a dose response effect on the lactate production, the maturation rate remained unaltered. It was observed that NADPH inhibited the activity of the G6PDH and the addition of NADP increased the activity of the enzyme (P < 0.05). To verify the effect of the physiological modulators on the COC pentose phosphate pathway (PPP), the proportion of colourless oocytes evaluated by brilliant cresyl blue (BCB) and the maturation rate were carried out. In presence of NADPH an inhibition (P < 0.05) on PPP and maturation rate was observed. On the other hand, NADP had no effect on PPP activity and maturation rate. The present study shows that the regulation of key enzymes of glucose metabolism in bovine COCs are regulated mainly by the energetic charge and the redox status. We also reported a tight relation between the activity of the PFK 1 and G6PDH enzymes, glycolytic and PPP activities and the oocyte maturation process.

Regulation of key enzymes of glucose metabolism in bovine COCs

The aim of this work was to study the regulation of PFK 1 and G6PDH, two key enzymes involved in glucose metabolism in cumulus oocyte-complexes (COCs), and its relationship with the oocyte maturation process. It was observed that the activity of PFK 1 in the presence of ATP was inhibited whereas the addition of AMP increased the activity (P < 0.05). To verify the effect of the physiological modulators on the COC glycolytic pathway, the lactate production during IVM and the maturation rate were evaluated. In accordance with the enzymatic activity, the glycolytic activity evaluated by lactate production and the maturation rate diminished (P < 0.05) with the addition of ATP. While the AMP had a dose response effect on the lactate production, the maturation rate remained unaltered. It was observed that NADPH inhibited the activity of the G6PDH and the addition of NADP increased the activity of the enzyme (P < 0.05). To verify the effect of the physiological modulators on the COC pentose phosphate pathway (PPP), the proportion of colourless oocytes evaluated by brilliant cresyl blue (BCB) and the maturation rate were carried out. In presence of NADPH an inhibition (P < 0.05) on PPP and maturation rate was observed. On the other hand, NADP had no effect on PPP activity and maturation rate. The present study shows that the regulation of key enzymes of glucose metabolism in bovine COCs are regulated mainly by the energetic charge and the redox status. We also reported a tight relation between the activity of the PFK 1 and G6PDH enzymes, glycolytic and PPP activities and the oocyte maturation process.(AU)

Glycolysis and gluconeogenesis in the liver of catfish fed with different concentrations of proteins, lipids and carbohydrates / Glicólise e gliconeogênese no fígado de jundiá alimentado com diferentes concentrações de proteínas, lipídeos e carboidratos

The activities of enzymes from a number of metabolic pathways have been used as a tool to evaluate the best use of nutrients on fish performance. In the present study the catfish Rhamdia quelen was fed with diets containing crude protein-lipid-carbohydrate (%) as follows: treatment (T) T1: 19-19-44; T2: 26-15-39; T3: 33-12-33; and T4: 40-10-24. The fish were held in tanks of re-circulated, filtered water with controlled temperature and aeration in 2000L experimental units. The feeding experiment lasted 30 days. The following enzymes of the carbohydrate metabolism were determined: Glucokinase (GK), Phosphofructokinase 1 (PFK-1), Pyruvate kinase (PK), Fructose-1,6-biphosphatase 1 (FBP-1). The activities of 6 phosphogluconate dehydrogenase (6PGDH) and glucose 6 phosphate dehydrogenase (G6PDH) were also assayed. The influence of nutrient levels on the enzyme activities is reported. The increase of dietary protein plus reduction of carbohydrates and lipids attenuates the glycolytic activity and induces hepatic gluconeogenesis as a strategy to provide metabolic energy from amino acids. The fish performance was affected by the concentrations of protein, lipid and carbohydrates in the diet. The greatest weight gain was obtained in fish fed diet T4 containing 40.14% of crude protein, 9.70% of lipids, and 24.37% of carbohydrate, respectively.(AU)

As atividades de enzimas das vias metabólicas têm sido utilizadas como uma ferramenta para avaliar a melhor utilização dos nutrientes e o desempenho dos peixes. No presente estudo, o jundiá foi alimentado com rações contendo diferentes concentrações de proteína bruta, lipídeos e carboidratos (%), da seguinte forma: tratamento (T) T1: 19-19-44; T2: 26-15-39; T3: 33-12-33; e T4: 40-10-24. Os peixes foram mantidos em tanques de recirculação, com água filtrada, temperatura controlada e aeração em unidades experimentais de 2.000L. O período experimental foi de 30 dias. Foram aferidas as atividades das enzimas glicoquinase (GK), fosfofrutoquinase 1 (PFK-1), piruvato quinase (PK) e frutose-1,6-difosfatase (FBP-1). Também foram aferidas as atividades da 6-fosfogluconato desidrogenase (6PGDH) e glicose-6-fosfato desidrogenase (G6PDH) da via das pentoses. É relatado que níveis de nutrientes influenciam as atividades enzimáticas das vias metabólicas. No presente estudo, o aumento da proteína da dieta e a redução de hidratos de carbono e lipídeos reduziram a atividade glicolítica e induziram a gliconeogênese hepática como uma estratégia para fornecer energia pelos aminoácidos. O desempenho dos peixes foi afetado pelas concentrações de proteínas, lipídeos e carboidratos na dieta. O maior ganho de peso foi obtido em peixes alimentados com dieta T4 contendo 40,14% de proteína bruta, 9,70% de lipídeos, e 24,37% de carboidratos, respectivamente.(AU)

Glycolysis and gluconeogenesis in the liver of catfish fed with different concentrations of proteins, lipids and carbohydrates / Glicólise e gliconeogênese no fígado de jundiá alimentado com diferentes concentrações de proteínas, lipídeos e carboidratos

The activities of enzymes from a number of metabolic pathways have been used as a tool to evaluate the best use of nutrients on fish performance. In the present study the catfish Rhamdia quelen was fed with diets containing crude protein-lipid-carbohydrate (%) as follows: treatment (T) T1: 19-19-44; T2: 26-15-39; T3: 33-12-33; and T4: 40-10-24. The fish were held in tanks of re-circulated, filtered water with controlled temperature and aeration in 2000L experimental units. The feeding experiment lasted 30 days. The following enzymes of the carbohydrate metabolism were determined: Glucokinase (GK), Phosphofructokinase 1 (PFK-1), Pyruvate kinase (PK), Fructose-1,6-biphosphatase 1 (FBP-1). The activities of 6 phosphogluconate dehydrogenase (6PGDH) and glucose 6 phosphate dehydrogenase (G6PDH) were also assayed. The influence of nutrient levels on the enzyme activities is reported. The increase of dietary protein plus reduction of carbohydrates and lipids attenuates the glycolytic activity and induces hepatic gluconeogenesis as a strategy to provide metabolic energy from amino acids. The fish performance was affected by the concentrations of protein, lipid and carbohydrates in the diet. The greatest weight gain was obtained in fish fed diet T4 containing 40.14% of crude protein, 9.70% of lipids, and 24.37% of carbohydrate, respectively.(AU)

As atividades de enzimas das vias metabólicas têm sido utilizadas como uma ferramenta para avaliar a melhor utilização dos nutrientes e o desempenho dos peixes. No presente estudo, o jundiá foi alimentado com rações contendo diferentes concentrações de proteína bruta, lipídeos e carboidratos (%), da seguinte forma: tratamento (T) T1: 19-19-44; T2: 26-15-39; T3: 33-12-33; e T4: 40-10-24. Os peixes foram mantidos em tanques de recirculação, com água filtrada, temperatura controlada e aeração em unidades experimentais de 2.000L. O período experimental foi de 30 dias. Foram aferidas as atividades das enzimas glicoquinase (GK), fosfofrutoquinase 1 (PFK-1), piruvato quinase (PK) e frutose-1,6-difosfatase (FBP-1). Também foram aferidas as atividades da 6-fosfogluconato desidrogenase (6PGDH) e glicose-6-fosfato desidrogenase (G6PDH) da via das pentoses. É relatado que níveis de nutrientes influenciam as atividades enzimáticas das vias metabólicas. No presente estudo, o aumento da proteína da dieta e a redução de hidratos de carbono e lipídeos reduziram a atividade glicolítica e induziram a gliconeogênese hepática como uma estratégia para fornecer energia pelos aminoácidos. O desempenho dos peixes foi afetado pelas concentrações de proteínas, lipídeos e carboidratos na dieta. O maior ganho de peso foi obtido em peixes alimentados com dieta T4 contendo 40,14% de proteína bruta, 9,70% de lipídeos, e 24,37% de carboidratos, respectivamente.(AU)

NaCl stress impact on the key enzymes in glycolysis from Lactobacillus bulgaricus during freeze-drying

Abstract The viability of Lactobacillus bulgaricus in freeze-drying is of significant commercial interest to dairy industries. In the study, L.bulgaricus demonstrated a significantly improved (p < 0.05) survival rate during freeze-drying when subjected to a pre-stressed period under the conditions of 2% (w/v) NaCl for 2 h in the late growth phase. The main energy source for the life activity of lactic acid bacteria is related to the glycolytic pathway. To investigate the phenomenon of this stress-related viability improvement in L. bulgaricus, the activities and corresponding genes of key enzymes in glycolysis during 2% NaCl stress were studied. NaCl stress significantly enhanced (p < 0.05) glucose utilization. The activities of glycolytic enzymes (phosphofructokinase, pyruvate kinase, and lactate dehydrogenase) decreased during freeze-drying, and NaCl stress were found to improve activities of these enzymes before and after freeze-drying. However, a transcriptional analysis of the corresponding genes suggested that the effect of NaCl stress on the expression of the pfk2 gene was not obvious. The increased survival of freeze-dried cells of L. bulgaricus under NaCl stress might be due to changes in only the activity or translation level of these enzymes in different environmental conditions but have no relation to their mRNA transcription level.(AU)

RITMOS DIÁRIOS DO METABOLISMO E FISIOLOGIA DIGESTIVA DE LARVAS DE TILÁPIA (Oreochromis niloticus) EM DIFERENTES TEMPERATURAS

Para apresentar um bom desempenho, as larvas devem conseguir assimilar os nutrientes da dieta, e para isso é necessário, entre alguns fatores, a presença de enzimas digestivas e metabólicas. A temperatura da água, além de ser um dos principais sincronizadores do ritmo circadiano, também é um dos fatores ambientais que mais influenciam no desenvolvimento embrionário e larvário dos peixes. Ela pode influenciar na expressão enzimática de enzimas digestivas e metabólicas, secreção de hormônios, desempenho e sobrevivência dos animais. Os fatores ambientais apresentam variações cíclicas ao largo do dia. Entre essas variações, as altas temperaturas aparecem durante o dia e as baixas temperaturas durante a noite, formando assim um termociclo. Sendo assim, o objetivo desse estudo foi avaliar a influência de diferentes temperaturas (termociclo e constante) no comportamento fisiológico, e crescimento de larvas de tilápia do Nilo (Oreochromis nilóticos). Para isso, ovos de tilápia foram fecundados e colocados imediatamente em incubadoras em dois sistemas distintos. Um sistema com temperatura constante (CTE) de 28ºC e um com termociclo (TC) de 31ºC: 25ºC. Ambos os sistemas ligados em um sistema de recirculação de água com filtro mecânico e biológico. O fotoperíodo foi de 12:12 LD (claro/escuro), com luzes acesas às 9:00h (Zeitgeber 0). As larvas eram alimentadas em horários fixos, quatro vezes ao dia, como uma dieta semi purificada contendo 42% de proteína bruta e 4,100 Kcal/kg de energia bruta. Larvas com 4, 8 e 13 dias pós fertilização (dpf) foram coletadas em ambos os tratamentos, em um intervalo de 4 horas ao longo de um ciclo de 24 hora para posteriores análises. O nível de expressão genica relativa das enzimas, foi analisada por qPCR. O crescimento das larvas foi avaliado através do seu comprimento padrão (experimento 1). Experimento 1: As larvas mantidas em TC apresentam um maior crescimento (teste-t, p<0.05) quando comparadas com as larvas mantidas em CTE. Os ritmos diários (Cosinor, p <0,05) da expressão dos genes quimiotripsina, lipase, maltase, isomaltase, colecistoquinina (cck) e neuropeptídeo y (npy) foram demostrados para larvas com 13dpf mantidas em TC, com picos de expressões (acrofase p <0,05) próximos as fases de alimentação. Experimento 2: As larvas com 13 dpf, mantidas em um TC apresentam diferenças significativas (Cosinor, p <0,05), nos ritmos diários na expressão relativa do gene alanina aminotransferase (alt), aspartato aminotransferase (ast), enzima málica, glicose-6-fosfato deseidrogenase (g6pd), piruvato quinase (pk) e fosfofrutoquinase (pfk). Enquanto as larvas mantidas em CTE, com a mesma idade, não apresentam ritmos diários (Cosinor, p >0,05). Além disso, a expressão enzimática (p<0,05) das larvas mantidas em TC, apresentaram acrofase entre o período de alimentação. Podemos concluir que o termociclo melhora a maturação do sistema circadiano das larvas, o que melhora sua eficiência digestiva e metabólica. Estes resultados podem ser úteis para melhorar os protocolos usados na larvicultura de tilápias aumentando o desempenho da produção.

Larvae have to assimilate the nutrients from the diet to get adequate growth rates and, for that purpose, it is necessary, among other factors, the presence of digestive and metabolic enzymes. Water temperature is one of the main synchronizers of biological rhythms and one of the most influential environmental factors for fish embryonic and larval development. Water temperature can influence the expression of digestive and metabolic enzymes, hormone secretion, growth and survival of animals. The environmental factors display cyclic variations along the day. Among them, high water temperature occurs during the day and low temperatures during the night, generating the thermocycle. Therefore, the objective of the present research was to evaluate the influence of different temperature regimes (thermocycle vs. constant) on the physiology and growth of Nile tilapia (Oreochromis niloticus) larvae. For that purpose, tilapia eggs were fertilized and immediately placed in incubators in two different rearing systems: one system with constant temperature (CTE) and one system with a thermocycle of 31ºC: 25ºC. Both were water recirculation systems equipped with mechanical and biological filters. Photoperiod was set at 12:12 LD (light/dark), with lights on at 9:00h (Zeitgeber time 0 h). Larvae were fed at fixed times, four times a day, with a semipurified diet of 42% of crude protein and 4,100 Kcal/kg of gross energy. Larvae at 4, 8 and 13 days postfertilization (dpf) were collected from both temperature treatments every 4 h along a 24 h cycle for further analyses. The levels of relative expression of all genes were analyzed by qPCR. Larval growth was measured through their standard length (experiment 1). Experiment 1: Larvae maintained at TC showed higher growth (t-test, p <0.05) than larvae kept at CTE. Daily rhythms (Cosinor, p <0.05) in the expression of chymotrypsin, lipase, maltase, isomaltase, cholecystokinin (cck) and neuropeptide Y (npy) were shown in 13dpf larvae reared in TC, with expression peaks close to the feeding phase. Experiment 2: larvae at 13 dpf and maintained at TC showed significant rhythms (Cosinor, p <0.05) of relative expression of the genes alanine aminotransferase (alt), aspartate aminotransferase (ast), malic enzyme, glucose-6-phosphate dehydrogenase (g6pd), pyruvate kinase (pk) and phosphofructokinase (pfk). On the other hand, larvae reared at CTE at the same stage did not present daily rhythms (Cosinor, p >0.05). The acrophase of expression of the enzymes in larvae maintained at TC appeared around mealtime. Therefore, it can be concluded that, under a thermocycle, tilapia larvae improve the maturation of their circadian system, which would thus improve their digestive and metabolic efficiency. These results can be helpful for improving protocols used in tilapia 20emperatura20 and increasing production performance.

Relationship of mammalian erythrocyte enzymes and their intermediary compounds among selected animals / Correlação of enzimas eritrocitárias e seus compostos intermediários de mamíferos selecionados

As there are few comparative studies on mammalian red cell metabolism, it was decided to study the glycolytic enzyme activities aswell as related ones, and the metabolites adenosine-5´-triphosphate(ATP) and 2,3-bisphosphoglycerate (2,3-BPG). Mammalia representatives from Primates, Rodentia, Carnivora, Lagomorpha,Artyodactyla, Didelphimorphia and Xenarthra orders, obtained from Fundação Parque Zoológico de São Paulo and Centro de Bioterismoda Faculdade de Medicina da USP, were studied. The blood was collectedin EDTA and ACD, the red cells were washed in saline at 4o C, lysed1:20 in hemolysing solution by freeze-and-thaw, and the followingenzymes were assayed according to standard procedures: hexokinase,glucose-6-phosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase,phosphoglycerate kinase, monophosphoglycerate mutase, enolase,pyruvate kinase, lactate dehydrogenase, as well as 2,3-bisphosphoglycerate mutase, glucose-6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase activities at 37oC , and adenosine-5´-triphosphate and 2,3-bisphophoglycerate concentrations. Are markable variation among the studied species was observed.However, it was detected a significant positive correlation between the adenosine-5´-triphosphate concentrations and triose phosphate isomerase and 2,3-bisphosphoglycerate mutase activities, as well assignificant positive correlation between 2,3-bisphosphoglycerate concentration and 2,3-bisphosphoglycerate mutase activity in all studied species as a whole. Most of studied species exhibited a steady ATP concentration range between 4 and 6 μ moles.g Hb –1 except the Artiodactyla (Cervus elaphus) and Carnivora (Panthera leo, Leoparduspardalis, Canis Lupus and Chrysocyon brachyurus,) which presented values between 2 and 3 μ moles g Hb –1. However, the 2,3-BPG concentration showed remarkable variation among the studied species and among the orders.(AU)

Como há poucos estudos comparativos sobre o metabolismo eritrócitário dos mamíferos propôs-se estudar as atividades das enzimas glicolíticas, anexas e os metabólitos adenosina-5-trifosfato e2,3-difosfoglicerato. Foram estudados mamíferos das ordens Primata,Rodentia, Carnivora, Lagomorpha, Artiodactyla, Didelphimorphia eXenarthra oriundos da Fundação Parque Zoológico de São Paulo e Centro de Bioterismo da Faculdade de Medicina da USP. O sangue foicolhido em EDTA e ACD, os eritrócitos foram lavados em soluçãofisiológica a 4o C e hemolisados em solução hemolisante 1:20 porcongelamento e descongelamento e as atividades das seguintes enzimasforam determinadas de acordo com procedimentos padronizados:hexoquinase, glicose-6-fosfato isomerase , fosfofrutoquinase, aldolase,triose fosfato isomerase, gliceraldeído-3-fosfato desidrogenase,fosfoglicerato quinase, 2,3-difosfoglicerato mutase,monofosfogliceromutase, enolase, piruvato quinase, lactatodesidrogenase, bem como a glicose-6-fosfato desidrogenase, 6-fosfogluconato desidrogenase a 37ºC e os metabólitos intermediários2,3-difosfoglicerato e adenosina-5-trifosfato. As enzimas e os compostos intermediários estudados apresentaram grande variabilidade entre as espécies de mamíferos estudadas. Foi observada correlação positiva entre a atividade da triose fosfato isomerase e a 2,3-difosfoglicerato mutase e os teores de adenosina-5-trifosfato das espécies, bem como correlação positiva entre a 2,3-difosfogliceratomutase em relação ao 2,3-difosfoglicerato. Os teores de adenosina-5-trifosfato mantiveram-se dentro de uma faixa estável, ao redor de 4 a6 μ moles / gHb, com as exceções das espécies das ordens Carnivora(Panthera leo, Leopardus pardalis, Canis lupus and Chrysocyonbrachyurus) e Artiodactyla (Cervus elaphus), que exibiram 2 a 3 μmoles / g Hb. Já os valores da concentração de 2,3-difosfoglicerato apresentaram, por sua vez, variação considerável entre as espécies e asordens estudadas.(AU)