Sandra Costa Valle

Lactate metabolism in the muscle of the crab Chasmagnathus granulatus during hypoxia and post-hypoxia recovery

The present study showed that the lactate/glucose ratio in the hemolymph of Chasmagnathus granulatus maintained in normoxia (controls) was 4.9, suggesting that lactate is an important substrate for this crab. Periods of hypoxia are part of the biological cycle of this crab, and lactate is the main end product of anaerobiosis in this crab. Our hypothesis was that this lactate would be, therefore, used by gluconeogenic pathway or can be oxidized or excreted to the aquatic medium during hypoxia and post-hypoxia periods in C. granulatus. The concentrations of hemolymphatic lactate in animals in normoxia are high, and are used as an energy substrate. In hypoxia, muscle gluconeogenesis and excretion of lactate to the aquatic medium would contribute significantly in regulating the concentration of circulating lactate. Utilization of these pathways would serve the objective of maintaining the acid-base equilibrium of the organism. Muscle gluconeogenesis participates, during the recovery process, in metabolizing the lactate produced during the period of hypoxia. Lactate excretion to the external medium, was one of the strategies used to decrease the higher hemolymphatic lactate levels. However, oxidation of lactate in the muscle is not a main strategy used by this crab to metabolize lactate in the recovery periods.

Glycine, serine, and leucine metabolism in different regions of rat central nervous system.

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-14C]glycine, [2-14C]glycine, L-[3-14C]serine and L-[U-14C]leucine. We showed that the [1-14C]glycine oxidation to CO2 in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-14C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-14C]leucine oxidation was lower than the [1-14C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO2 in all structure studied. In adult rats brains, the highest oxidation of [1-14C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-14C]leucine was significantly higher than that from glycine in all regions and ages studied.

Study of developmental changes on hexoses metabolism in rat cerebral cortex

We have studied the developmental changes of glucose, mannose, fructose and galactose metabolism in rat cerebral cortex. As the animals aged, glucose, mannose and fructose oxidation to CO2 increased, whereas galactose oxidation decreased. Lipid synthesis from glucose and fructose also increased with age, that from mannose decreased and galactose did not change. Cytochalasin B, a potent non-competitive inhibitor of sodium-independent glucose transport, significantly impaired glucose, mannose and galactose metabolism, but had no effect on fructose metabolism. Both galactose or fructose did not change, whereas mannose declined the glucose metabolism. Glucose decreased fructose, galactose and mannose metabolism. Our results show that besides glucose, the metabolism of mannose, galactose and fructose present developmental changes from fetal to adult age, and reinforce the literature data indicating that mannose and galactose are transported by glucose carriers, while fructose is not.

Seasonal variation in glucose and neutral amino acid uptake in the estuarine crab Neohelice granulata.

This study investigates the mechanisms of glucose and amino acid transport in gills and jaw muscle of N. granulata collected from an estuarine natural population. The physicochemical parameters of the estuarine environment and of this crustaceans hemolymph were measured during different seasons of the year. In summer, the lagoon water osmolality increased (5-6 times), and hemolymph osmolality decreased. In fall, water pH increased, whereas hemolymph pH decreased markedly. In all seasons, 2-deoxi glucose (DG) uptake in gills was significantly higher than 3-O methyl-glucose (MG) uptake. Phloretin reduced DG uptake in gills; phloretin and phlorizin did not affect MG uptake in this organ. DG and MG uptake was highest in gills during spring and summer. In jaw muscle, MG uptake in winter and spring was higher than DG uptake. In fall, gill methyl aminoisobutyric acid (MeAIB) uptake increased. In jaw muscle, MeAIB uptake was higher in spring. The observed changes in glucose uptake and in the type of glucose and amino acid transporter used in gills and muscle appear to be strategies used by N. granulata to minimize seasonal oscillations in the environmental parameters of their estuarine habitat.

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-14C]leucine and [1–14C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-14C]leucine and [1–14C]glycine and from l-[U-14C]leucine, respectively; glucose, β-OH-butyrate and lactate stimulated lipid synthesis from l-[U-14C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO2 as well as lipid synthesis from l-[U-14C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-14C]leucine and [1–14C]glycine; glutamine also markedly decreased the oxidation of l-[U-14C]leucine and [1–14C]glycine in 10–day-old and adult rats.

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata : Effects of osmotic stress

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG.

Características de ligação da insulina no tecido muscular canino: efeitos da fase do ciclo estral

As flutuacoes hormonais durante as diferentes fases do ciclo estral sao uma causa importante de resistencia insulinica em femeas caninas, e poucas informacoes sao conhecidas sobre defeitos na ligacao da insulina ao seu receptor, ou defeitos pos-receptor associados com resistencia a insulina em caes. Para avaliar as caracteristicas da ligacao insulina-receptor no tecido muscular de cadelas durante o ciclo estral, dezessete pacientes foram utilizadas no estudo (seis em anestro, cinco em estro e seis em diestro). Um teste de tolerância a glicose intravenosa (IVGTT) foi realizado em todas as pacientes por meio da infusao de 1mL/kg de uma solucao de glicose 50% (500mg/kg), com coletas de sangue para determinacao de glicemia nos tempos 0, 3, 5, 7, 15, 30, 45 e 60 minutos da injecao de glicose. Amostras de tecido muscular foram coletadas durante ovariohisterectomia, imediatamente congeladas em nitrogenio liquido, e posteriormente armazenadas a -80°C ate a preparacao das membranas por meio de homogeneizacao e centrifugacao sequencial. Para os experimentos de ligacao hormonio-receptor, as membranas foram incubadas na presenca de 20.000cpm de 125I-insulina humana, e concentracoes crescentes de insulina regular humana nao marcada para saturacao fria. O IVGTT nao mostrou diferencas entre as pacientes em diferentes fases do ciclo estral com relacao a glicemia basal, ou na resposta glicemica apos infusao de glicose nos tempos estudados. Dois sitios de ligacao da insulina, um de alta-afinidade, e outro de baixa afinidade, foram detectados pela analise de Scatchard, e diferencas significativas foram detectadas na constante de dissociacao (Kd1) e capacidade de ligacao maxima (Bmax1) dos sitios de ligacao de alta-afinidade. O Kd1 para o grupo anestro (6,54±2,77nM/mg de proteina) foi menor (P<0,001) que os Kd1 dos grupos estro (28,54±6,94 nM/mg de proteina) e diestro (15,56±3,88nM/mg de proteina). Os Bmax1 dos grupos estro (0,83±0,42nM/mg de proteina) e diestro (1,24±0,24nM/mg de proteina) tambem foram maiores que os valores encontrados no grupo anestro (0,35±0,06nM/mg de proteina). Estes resultados demonstram uma modulacao das caracteristicas de ligacao da insulina nas diferentes fases do ciclo estral em caes, evidenciando uma menor afinidade de ligacao da insulina ao seu receptor no tecido muscular durante o estro e diestro. Contudo, esta menor afinidade de ligacao hormonio-receptor e compensada por uma maior capacidade de ligacao, o que fica tambem evidenciado pela ausencia de diferencas na resposta glicemica das pacientes apos um desafio com glicose por via endovenosa.