Alberto M. Vargas

Evolution of gluconeogenic enzyme activities during starvation in liver and kidney of the rainbow trout (Salmo gairdneri)

1. There was a general increase in the activities of enzymes involved in gluconeogenesis in liver and kidney of rainbow trout, Salmo gairdneri, during the second month of starvation. 2. The need of gluconeogenesis during the first month of the starvation period was probably minimal because of the utilization of liver glycogen as a source of blood glucose. 3. The decline of fat was more pronounced than that of protein total content in absolute values, suggesting that lipid reserved were the main sources of energy during starvation.

DIFFERENTIAL BEHAVIOUR OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE IN TWO MORPHOLOGICAL FORMS OF TRYPANOSOMA CRUZI

1. Glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49) of two morphological forms of Trypanosoma cruzi, epimastigotes and metacyclics, are reported. 2. The kinetic behaviour and some of the kinetic parameters of the enzyme in both forms were studied. The enzymes showed a simple Michaelis-Menten kinetic. 3. The activity in epimastigote forms was alway higher than the metacyclic ones. At subsaturating concentrations of substrate was almost 10-fold higher, whereas at saturating concentrations was about 2-fold higher. 4. In epimastigote forms the specific activity and Km values, at pH 7.5 and 37 degrees C, was found to be 142 mUnits x mg-1 of protein and 0.23 mM, respectively. 5. In the same conditions, the specific activity and Km values in metacyclic forms was 75 mUnits x mg-1 of protein and 1.06 mM, respectively. 6. A possible role in the carbohydrate metabolism of glucose 6-phosphate dehydrogenase in both forms of Trypanosoma cruzi is discussed.

Hormonal effects on the liver glucose metabolism in rainbow trout (Salmo gairdneri).

1. Glucagon, adrenaline and dibutyril cyclic AMP increased the release of glucose to the medium during incubation of liver slices from rainbow trout (Salmo gairdneri) while insulin had no effect. 2. Glycogen content decreased only slightly after cyclic AMP addition and even increased in the presence of glucagon and adrenaline. Consequently, the release of glucose was due mainly to gluconeogenesis. 3. This is corroborated by the reduction of glucose liberation in presence of alpha-cyanocinnamate, an inhibitor of gluconeogenesis.

Internalization of the Receptor for Advanced Glycation End Products (RAGE) is Required to Mediate Intracellular Responses

To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE-EGFP protein is expressed at the plasma membrane of CHO-k1 and Neuro-2a (N2a) cells and retains the capacity to bind Texas Red-labelled advanced glycation end products (AGEs). AGEs addition to the cell cultures induced a change in the subcellular distribution of the fluorescent RAGE-EGFP protein compatible with an internalization of the AGEs-RAGE complex. Furthermore, while N2a cells expressing the RAGE-EGFP showed an increase in ERK1/2 phosphorylation and NF-kappaB DNA binding in response to AGEs, pre-incubation with dansyl-cadaverine or phenylarsine oxide, inhibitors of receptors internalization, blocked the activation of ERKs and other intracellular responses mediated by AGEs. These results suggest that internalization plays a key role in the signal transduction mediated by RAGE.

Modulation of glucose transporters in rat diaphragm by sodium tungstate

Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. We examined the effects of 6 weeks of oral administration of tungstate on glucose transporters (GLUT) in streptozotocin‐induced diabetic rat diaphragm. Diabetes decreased GLUT4 expression while tungstate treatment normalized not only GLUT4 protein but also GLUT4 mRNA in the diabetic rats. Furthermore, treatment increased GLUT4 protein in plasma and internal membranes, suggesting a stimulation of its translocation to the plasma membrane. Tungstate had no effect on healthy animals. There were no differences in the total amount of GLUT1 transporter in any group. We conclude that the normoglycemic effect of tungstate may be partly due to a normalization of the levels and subcellular localization of GLUT4, which should result in an increase in muscle glucose uptake.

The glucose-lowering agent sodium tungstate increases the levels and translocation of GLUT4 in L6 myotubes through a mechanism associated with ERK1/2 and MEF2D.

Aims/hypothesisThe aim of this study was to investigate the action of the glucose-lowering compound sodium tungstate on glucose transport in muscle myotubes and to unravel the molecular events underlying the effects observed.MethodsWe studied the effects of tungstate on 2-deoxy-d-glucose uptake, levels and translocation of the glucose transporters GLUT4 and GLUT1, and Glut4 (also known as Slc2a4) promoter activity. We also measured the modifications of individual components of the signalling pathways involved in the effects observed.ResultsTungstate increased 2-deoxy-d-glucose uptake in differentiated L6 myotubes through an increase in the total amount and translocation of GLUT4 transporter. The effects on glucose uptake were additive to those of insulin. Tungstate activated transcription of the Glut4 promoter, as shown by an increase in Glut4 mRNA, and by a promoter reporter assay. The assay of deletions of the Glut4 promoter indicated that the effect of tungstate is mediated by the myocyte enhancer factor 2 (MEF2)-binding domain. Accordingly, MEF2 levels and DNA binding activities were increased in response to the treatment. Tungstate-induced glucose uptake and GLUT4 transcriptional activation were dependent on the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), while no changes were observed in the phosphorylation state of the β subunit of the insulin receptor, in the phosphatidylinositol 3-kinase pathway or in the activation of 5′AMP-activated protein kinase.Conclusions/interpretationTungstate activates glucose uptake in myotubes through a novel ERK1/2-dependent mechanism. This effect is exerted by an increase in the content and translocation of the GLUT4 transporter. This is the first report of a glucose-lowering compound activating Glut4 transcription through an ERK1/2-dependent increase in MEF2 levels.

Involvement of gluconeogenesis in the hyperglycemia induced by glucagon, adrenaline and cyclic AMP in rainbow trout (Salmo gairdneri)

Abstract 1. 1. The injection of adrenaline or dibutyril cyclic AMP to rainbow trout, Salmo gairdneri was followed by an increase in blood glucose and lactate. 2. 2. The injection of glucagon was followed by hyperglycemia without increase in blood lactate. 3. 3. None of these treatments produced significant alterations in liver glycogen. 4. 4. The simultaneous injection of dibutyril cyclic AMP and α-cyano-hydroxy-cinnamate resulted in a substantially smaller increase in blood glucose concentration than the injection of dibutyril cyclic AMP alone. 5. 5. These results show that hyperglycemia was due to gluconeogenesis under these circumstances, confirming the key role of this process in the trout.

Increased Diaphragm Expression of GLUT4 in Control and Streptozotocin-Diabetic Rats by Fish Oil-Supplemented Diets

Dietary fat intake influences plasma glucose concentration through modifying glucose uptake and utilization by adipose and skeletal muscle tissues. In this paper, we studied the effects of a low-fat diet on diaphragm GLUT4 expression and fatty acid composition in control and streptozotocin-induced diabetic rats. Control as well as diabetic rats were divided into three different dietary groups each. Either 5% olive oil, 5% sunflower oil, or 5% fish oil was the only fat supplied by the diet. Feeding these low-fat diets for 5 wk induced major changes in fatty acid composition, both in control and in diabetic rats. Arachidonic acid was higher in diabetic olive and sunflower oil-fed rats with respect to fish oil-fed, opposite to docosahexaenoic acid which was higher in diabetic fish oil-fed rats with respect to the other two groups. Animals receiving a fish oil diet had the lowest plasma glucose concentration. GLUT4 expression in diaphragm, as indicated by GLUT4 protein and mRNA, is modulated both by diabetes and by diet fatty acid composition. Diabetes induced a decrease in expression in all dietary groups. Plasma glucose levels correlated well with the increased amount of GLUT4 protein and mRNA found in fish oil-fed groups. Results are discussed in terms of the influence that arachidonic and n−3 polyunsaturated fatty acids may exert on the transcriptional and translational control of the GLUT4 gene.

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney

The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period

Postharvest changes in total soluble solids and tissue pH of cherimoya fruit stored at chilling and non-chilling temperatures

SummaryTotal soluble solids (TSS) and pH were determined in cherimoya fruit (Annona cher- imola Mill.) stored at several temperatures. TSS increased from about 8.3° Brix to 22° Brix during 4 d of ripening at 22°C. At the same time fruit pulp pH declined from 6.2 to 4.3. Similar values in TSS and pH, were not reached until the tenth day at 12°C. Fruit stored at 4°C underwent a slower increase in TSS and the decrease in pH was less than 1 unit. Storage at 1°C for 27 d did not modify pH, and TSS increased by only 2° Brix. Chilling injury was dependent on the length and temperature of storage. Fruit rewarmed to 22°C after storage for 11 d at 4°C underwent changes in TSS and pH similar to those found in fruit placed directly into 22°C air; on the contrary, pH was not modified when the chilling storage was for 15 d. However, rewarming of fruit previously stored at 1°C resulted in a slight increase in TSS and little change in pH.