Forms of application of nitrogen in different stages of the development of soybean culture / Formas da aplicação de nitrogênio em diferentes estádios do desenvolvimento da cultura da soja

In Brazil, inoculation with Bradyrhizobium japonicum and B. elkanii strains is the main form of Nitrogen (N) supply to soybean, but there are reports of the use of mineral N as a complement to biological fixation. The objective was to evaluate the effect of different forms and times of N application on the development and yield of soybean. The treatments include a control without N application and in all other treatments 10 kg ha-1 of N was applied, altering the time (V2, V4, R1 or R2) and the application form, namely, at sowing, broadcast on topdressing or by leaf spraying. The application of N provided an increase in leaf area, dry mass of shoot, number of nodules and dry mass of nodules in treatments with application of N at sowing and N at V2 on topdressing. For plant height, the treatment with N at V2 by leaf spraying was superior compared to control. The weight of one thousand seeds was not affected by N application. For grain yield the treatments with application of N by leaf spraying in V2 and R1 were highlighted. The greatest increase in grain yield on soybean is reached with leaf spraying at V2 stage

Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses

Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives: This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology: In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results: In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion: Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans

Participação de estoques intracelulares de Ca2+ no mecanismo de secreção de insulina : efeito do anestesico local tetracaina

Orientador: Antonio Carlos BoscheroTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Foi realizado estudo do efeito da tetracaína sobre o efluxo de 45Ca, a concentração de Ca2+ citoplasmático, [Ca2+]i, e a secreção de insulina em ilhotas pancreáticas e células B isoladas. Na ausência de Ca2+ externo, tetracaína (0,12,0 mM) aumentou, de maneira dose-dependente, o efluxo de 45Ca de ilhotas isoladas. Tetracaína não afetou o aumento do efluxo de 45.ca causado por 50 mM de K+ ou pela associação de carbacol (Cch - 0,2 mM) e 50 mM de K+. Tetracaína aumentou permanentemente a [Ca2+]i em células B isoladas em meio livre de Ca2+ e acrescido de 2,8 mM de glicose e 25 µM D-600 (metoxiveparamil). Este efeito também foi observado na presença de 10 mM de cafeína ou 1 µM de tapsigargina. Em presença de 16,7 mM de glicose, tetracaína aumentou de maneira transitória a secreção de insulina das ilhotas perfundidas, tanto na ausência quanto na presença de Ca2+ externo. Estes dados indicam que tetracaína mobiliza Ca2+ de um estoque insensível à tapsigargina e estimula a secreção de insulina na ausência de Ca2+ extracelular. O aumento do efluxo de 45Ca causado pelas altas concentrações de K+ e pelo Cch indica que tetracaína não interfere nos estoques sensíveis a cátions ou ao IP3Abstract: The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i and insulin secretion in isolated pancreatic islets and B-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) dose-dependently increased the 45Ca efflux from isolated islets. Tetracaine did not affect the increase in 45Ca efflux caused by 50 mM K+ or by the association of carbachol (Cch - 0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated B-cells in Ca2+ -free medium enriched with 2.8 mM glucose and 25 µM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 µM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perifused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by Cch indicates that tetracaine did not interfere with a cation or IP3-sensitive Ca2+ pool in B-cellsDoutoradoFisiologiaDoutor em Ciência

Involvement of the cholinergic pathway in glucocorticoid-induced hyperinsulinemia in rats

Aims: We investigated the contribution of the cholinergic nervous system to dexamethasone-induced insulin resistance and hyperinsulinemia in rats.Methods: Seventy-day-old Wistar male rats were distributed in groups: control (CTL), vagotomized (VAG), and sham operated (SHAM). on the 90th day of life, half of the rats were treated daily with 1 mg/kg of dexamethasone for 5 days (CTL DEX, VAG DEX, and SHAM DEX).Results: In the presence of 8.3 mM glucose plus 100 mu M carbachol (Cch), isolated islets from CTL DEX secreted significantly more insulin than CTL. Cch-enhancement of secretion was further increased in islets from VAG CTL and VAG DEX than SHAM CTL and SHAM DEX, respectively. In CTL DEX islets, M3R and PLC beta 1 and phosphorylated PKC alpha, but not PKC alpha, protein content was significantly higher compared with each respective control. In islets from VAG DEX, the expression of M3R protein increased significantly compared to VAG CTL and SHAM DEX. Vagotomy per se did not affect insulin resistance, but attenuated fasted and fed insulinemia in VAG DEX, compared with SHAM DEX rats.Conclusion: These data indicate an important participation of the cholinergic nervous system through muscaric receptors in dexamethasone-induced hyperinsulinemia in rats. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Higher Insulin Sensitivity and Impaired Insulin Secretion in Cachetic Solid Ehrlich Tumour-Bearing Mice

Insulin secretion is mainly regulated by blood glucose concentration. On the other hand, changes in peripheral insulin sensitivity induce compensatory adaptations in pancreatic beta-cells to maintain normoglycaemia. Tumour presence causes dramatic alterations in glucose homeostasis and insulin secretion because of the high glucose consumption by the tumour cells. Here, we investigated insulin secretion in solid Ehrlich tumour-bearing mice in association with cachexia. For that, male adult Swiss mice were subcutaneously inoculated with solid Ehrlich tumour cells and sacrificed at 14 days after tumour implantation (SET), while control mice received saline alone (CTL). Insulin secretion, following different stimuli, glucose tolerance, and insulin sensitivity as well as the expression of key proteins involved in insulin secretion was assessed. The SET group showed decreased glycaemia, insulinaemia, hepatic glycogen and body weight, and increased plasma free fatty acids and triglycerides, characteristics of cancer cachexia. A very interesting finding in this study was the development of higher glucose tolerance and insulin sensitivity in SET group. The dose-response curve of insulin secretion to increasing glucose concentrations (2.8-22.2 mM) showed an EC50 of 10 mM glucose for CTL mice and 13 mM glucose for SET mice. Insulin secretion was significantly reduced in SET islets at 30 mM KCl, 100 M carbachol, 20 mM arginine, and 20 mM leucine. Moreover, AKT, PKA, PKC, and AchRM3 expressions were reduced by 17 % to 24 % in SET animals. These results, mainly the augmented insulin sensitivity, show that SET is an interesting model to study alterations in pancreatic function and carbohydrate metabolism in cancer cachexia.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Functional alterations in endocrine pancreas of rats with different degrees of dexamethasone-induced insulin resistance

Objectives: We have analyzed the peripheral insulin and glucose sensitivity in vivo, and islet function ex vivo in rats with different degrees of insulin resistance induced by dexamethasone (DEX).Methods: Dexamethasone, in the concentrations of 0.1 (DEX 0.1), 0.5 (DEX 0.5), and 1.0 mg/kg body weight (DEX 1.0) was administered daily, intraperitoneally, to adult Wistar rats for 5 days, whereas controls received saline.Results: Dexamethasone treatment induced peripheral insulin resistance in a dose-dependent manner. At the end of the treatment, only DEX 1.0 rats showed significant increase of postabsorptive blood glucose and serum triglycerides, and nonesterified fatty acids levels. Incubation of pancreatic islets in increasing glucose concentrations (2.8-22 mM) led to an augmented insulin secretion in all DEX-treated rats. Leucine, carbachol, and high KCl concentrations induced the insulin release in DEX 0.5 and DEX 1.0, whereas arginine augmented secretion in all DEX-treated groups.Conclusions: We demonstrate that in DEX 0.5 and, especially in DEX 0.1 groups, but not in DEX 1.0, the adaptations that occurred in the endocrine pancreas are able to counteract metabolic disorders (glucose intolerance and dyslipidemia). These animal models seem to be interesting approaches for the study of degrees of subjacent effects that may mediate type 2 diabetes (DEX 1.0) and islet function alterations, without collateral effects (DEX 0.1 and DEX 0.5)

Cellular changes in the prostatic stroma of glucocorticoid-treated rats

Glucocorticoid hormones (GCs) have been widely used for the treatment of prostate cancer because of their inhibitory property against tumour growth. However, their mechanism of action in the prostate has received little attention. Excess GCs can lead to peripheral insulin resistance resulting in hyperglycaemia and hyperinsulinaemia. Insulin plays an important role as a cellular stimulant and high levels are related to low levels of androgens. Our objective has been to describe the effects of insulin resistance induced by dexamethasone treatment on the morphology of rat ventral prostate. Mate adult Wistar rats received daily intraperitoneal injections of dexamethasone or saline for five consecutive days after which the rats were killed and the ventral prostate was removed, weighed and prepared for conventional and transmission electron microscopy (TEM). Dexamethasone treatment resulted in atrophy and decreased proliferative activity of prostatic epithelial cells. TEM analysis revealed changes in the epithelium-stroma interface, with some interruptions in the basement membrane. Fibroblasts showed a secretory phenotype with dilated endoplasmic reticulum. Smooth muscle cells exhibited a contractile pattern with 50% atrophy, an irregular membrane and twisted nuclei. Mitochondrial alterations, such as enlarged size and high electron density in the mitochondrial matrix, were also detected in smooth muscle cells. Insulin resistance induced by dexamethasone is thus associated with epithelial atrophy similar to that described for diabetic rats. However, GCs are responsible for morphological changes in the stromal cell population suggesting the activation of fibroblasts and atrophy of the smooth muscle cells

Metabolic parameters of rats prone to display wild-running of the audiogenic crisis and fighting induced by REM-sleep deprivation

Total sleep or selective REM-sleep deprivation (SD) increases aggressiveness. Rats that display fighting when submitted to sleep-deprivation are those that manifest wild running (WR), a panic-like flight that precedes tonic-clonic seizures induced by intense acoustic stimulation (audiogenic epilepsy). The incidence of WR-sensitive rats in the colonies around the world may reach 20%, which makes important to know the other characteristics of these animals for both sleep and other kinds of experimental research. Based on the report of their reduced body weight, we investigated some glucose metabolism parameters. Methods: Adult male Wistar rats were submitted to high-intensity acoustic stimulation (112dB, 60s) and then classified as WR-sensitive or WR-resistant rats. Glycemia and insulinemia, measured by glucometer and radioimmunoassay, respectively, and body weight were analyzed during three months. At the end of this period, intraperitoneal glucose and insulin tolerance tests were performed, and hepatic fat and glycogen content were also determined. Results: WR-sensitive rats showed proportionally less gain of body mass compared to the resistant ones (p<0.05) in initial measurements, but this tendency did not sustain thereafter. Interestingly, fasting glycemia was significantly lower in the WR-sensitive group throughout the experiment, compared to WR-resistant rats. No significant differences between groups were found in insulinemia, peripheral glucose and insulin tolerance, and hepatic glycogen. However, WR-sensitive rats increased hepatic fat content significantly after a 12-hour fasting. Conclusions: Slow weight gain, decreased fasting glycemia and hepatic steatosis suggest a possible accelerated energetic metabolism or low resistance to food deprivation, which could partially explain the reduced body weight of WR-sensitive rats.A privação total ou seletiva de sono REM aumenta a agressividade. Ratos que manifestam brigas quando submetidos a tal priva- ção são os mesmos que manifestam a corrida selvagem (WR, wild running), uma fuga panicoforme que precede a convulsão tônico-clônica induzida pela estimulação sonora intensa (epilepsia audiogênica). A incidência de ratos sensíveis à WR nas colônias ao redor do mundo pode alcançar 20%, e isso torna importante conhecer as outras características desses animais tanto nos estudos de sono como em outras espécies de investigação experimental. Baseados no relato de que esses animais têm peso corporal reduzido, foram investigados alguns parâ- metros do metabolismo da glicose. Métodos: Ratos Wistar machos adultos foram submetidos à estimulação acústica de alta intensidade (112 dB, 60s) e classificados como propensos ou resistentes à WR. A glicemia e insulinemia foram medidas por glicosímetro e por radioimunoensaio, respectivamente. O peso corporal foi avaliado por mais de três meses. Ao final do período, os testes de tolerância à glicose e à insulina administrada intraperitonealmente foram efetivados paralelamente às determinações do conteúdo hepático de gorduras e de glicogênio. Resultados: Os ratos propensos à WR mostraram ganhar relativamente menos massa corporal em relação aos animais resistentes (p<0,05) no início das observações; entretanto, esta tendência não se manteve subsequentemente. De maneira interessante, a glicemia em jejum foi significativamente menor no grupo de animais propensos à WR quando comparados aos animais resistentes. Não foram encontradas diferenças significativas entre os grupos no tocante à insulinemia, tolerância à glicose e à insulina administrada perifericamente, assim como no glicogênio hepático. Contudo, os ratos propensos à WR aumentaram significantemente o conteúdo de gordura hepática após 12 horas de jejum. Conclusões: O ganho lento de peso, a redução das glicemias de jejum e a esteatose hepática sugerem a possibilidade de um metabolismo energético acelerado ou baixa resistência à privação alimentar, o que pode explicar parcialmente o peso corporal reduzido em ratos propensos à WR.Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP

Dexamethasone-induced insulin resistance is associated with increased connexin 36 mRNA and protein expression in pancreatic rat islets

Augmented glucose-stimulated insulin secretion (GSIS) is an adaptive mechanism exhibited by pancreatic islets from insulin-resistant animal models. Gap junction proteins have been proposed to contribute to islet function. As such, we investigated the expression of connexin 36 (Cx36), connexin 43 (Cx43), and the glucose transporter Glut2 at mRNA and protein levels in pancreatic islets of dexamethasone (DEX)-induced insulin-resistant rats. Study rats received daily injections of DEX (1 mg/kg body mass, i.p.) for 5 days, whereas control rats (CTL) received saline solution. DEX rats exhibited peripheral insulin resistance, as indicated by the significant postabsorptive insulin levels and by the constant rate for glucose disappearance (K-ITT). GSIS was significantly higher in DEX islets (1.8-fold in 16.7 mmol/L glucose vs. CTL, p < 0.05). A significant increase of 2.25-fold in islet area was observed in DEX vs. CTL islets (p < 0.05). Cx36 mRNA expression was significantly augmented, Cx43 diminished, and Glut2 mRNA was unaltered in islets of DEX vs. CTL (p < 0.05). Cx36 protein expression was 1.6-fold higher than that of CTL islets (p < 0.05). Glut2 protein expression was unaltered and Cx43 was not detected at the protein level. We conclude that DEX-induced insulin resistance is accompanied by increased GSIS and this may be associated with increase of Cx36 protein expression