Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells

Interleukin-1 (IL-1) may be a mediator of β-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor κB (NF-κB), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-κB activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1β, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 μM) prevented the increase in nitrite production by rat islets exposed to IL-1β for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1β for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1β, tumor necrosis factor-α and interferon-γ). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-β but failed to block IL-1β-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1β-induced NF-κB activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic β-cells, an effect not associated with inhibition of NF-κB activation.

Long-term effect of prolactin treatment on glucose-induced insulin secretion in cultured neonatal rat islets

Insulin secretion and 45SCa2+ uptake and efflux were studied in neonatal rat islets maintained in culture for 7 or 19 days in the absence or presence of prolactin (PRL). Insulin secretion in response to glucose (G), leucine (Leu), arginine (Arg) and carbachol (Cch) was augmented after 7 and 19 days in culture, compared to basal secretion (G 2.8 mM), in both PRL- treated and control islets. However, the increase in insulin secretion induced by the above secretagogues was higher in islets cultured in the presence of PRL for 19 days. In PRL-treated islets, the 45Ca2+ content after a 5 min incubation in the presence of G, Leu, Arg and Cch was significantly higher than the control only in islets cultured for 19 days. Except with Arg, the 45Ca2+ uptake in PRL-treated islets after a 90 min incubation was also significantly higher than the control only in islets cultured for 19 days. Finally, Leu-induced alterations in the 45Ca2+ efflux were higher in PRL-treated than in control islets cultured for 7 or 19 days. In the absence of external Ca2+, the reduction in 45Ca2+ efflux induced by glucose was also significantly higher in PRL-treated than in control islets. This effect was slightly potentiated after 19 days in culture. These data further support the hypothesis that PRL treatment enhances maturation of the secretory mechanism in neonatal islets. This effect can be potentiated even more if the treatment is prolonged.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Effects of sublethal doses of imidacloprid in malpighian tubules of africanized Apis mellifera (Hymenoptera, Apidae)

In Brazil, imidacloprid is a widely used insecticide on agriculture and can harm bees, which are important pollinators. The active ingredient imidacloprid has action on the nervous system of the insects. However, little has been studied about the actions of the insecticide on nontarget organs of insects, such as the Malpighian tubules that make up the excretory and osmoregulatory system. Hence, in this study, we evaluated the effects of chronic exposure to sublethal doses of imidacloprid in Malpighian tubules of Africanized Apis mellifera. In the tubules of treated bees, we found an increase in the number of cells with picnotic nuclei, the lost of part of the cell into the lumen, and a homogenization of coloring cytoplasm. Furthermore, we observed the presence of cytoplasmic vacuolization. We confirmed the increased occurrence of picnotic nuclei by using the Feulgan reaction, which showed the chromatin compaction was more intense in the tubules of bees exposed to the insecticide. We observed an intensification of the staining of the nucleus with Xylidine Ponceau, further verifying the cytoplasmic negative regions that may indicate autophagic activity. Additionally, immunocytochemistry experiments showed TUNEL positive nuclei in exposed bees, implicating increased cell apoptosis after chronic imidacloprid exposure. In conclusion, our results indicate that very low concentrations of imidacloprid lead to cytotoxic activity in the Malpighian tubules of exposed bees at all tested times for exposure and imply that this insecticide can alter honey bee physiology. © 2013 Wiley Periodicals, Inc.

Mechanism and effect of esculetin in an experimental animal model of inflammatory bowel disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 μM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 μM), and E-64 (IC 50 3 μM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14+ human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK. © FASEB.

Neutrophil dysfunction varies with the stage of canine visceral leishmaniosis

Canine visceral leishmaniosis (CVL) causes a dependent-stage alteration in neutrophil oxidative metabolism. When production of reactive oxygen species (ROS) exceeds the antioxidant capacity of neutrophils, apoptosis is triggered, impairing the viability and function of these cells, which can predispose dogs to infection. However, the uremic condition observed in late-stage CVL can also alter the viability and function of human neutrophils. To more clearly understand this relationship, the apoptosis rate and oxidative metabolism of neutrophils from control dogs (n= 20) were compared to dogs in moderate (n= 15) and very severe (n= 15) stage CVL, classified according to LeishVet Consensus. To assess neutrophil oxidative metabolism, superoxide production was measured using the nitroblue tetrazolium reduction test (NBT) in isolated neutrophils. The apoptosis rate of neutrophils was estimated using the morphological method. Moderate-stage dogs presented increased superoxide production, while dogs with very severe stage CVL presented decreased superoxide production and an increase neutrophil apoptosis rate. Leishmaniosis causes differential neutrophil dysfunction according to disease stage. In moderate stage CVL, increased superoxide production is observed with no change in neutrophil viability. However, in very severe stage CVL, decreased superoxide production and increased apoptosis occur associated with uremia. © 2013 Elsevier B.V.

Estruturação ex-vivo de vasos sanguíneos a partir da diferenciação de células tronco de coelhos

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Dopplervelocimetria da artéria umbilical e controle glicêmico materno como marcadores de alterações vasculares e apoptóticas placentárias

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Age- and gender-related changes in glucose homeostasis in glucocorticoid-treated rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polimorfismos GSTM1, GSTT1 e GSTP1 da enzima Glutationa S-transferase como fatores moduladores do fenótipo na anemia falciforme

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Apoptose relacionada à infecção in vitro por herpesvírus bovinos tipo 1 e 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pharmacokinetics and pharmacodynamics of the glucagon-like peptide-1 analog liraglutide in healthy cats

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that induces glucose-dependent stimulation of insulin secretion while suppressing glucagon secretion. Glucagon-like peptide-1 also increases beta cell mass and satiation while decelerating gastric emptying. Liraglutide is a fatty-acid derivative of GLP-1 with a protracted pharmacokinetic profile that is used in people for treatment of type II diabetes mellitus and obesity. The aim of this study was to determine the pharmacokinetics and pharmacodynamics of liraglutide in healthy cats. Hyperglycemic clamps were performed on days 0 (HGC) and 14 (LgHGC) in 7 healthy cats. Liraglutide was administered subcutaneously (0.6 mg/cat) once daily on days 8 through 14. Compared with the HGC (mean +/- standard deviation; 455.5 +/- 115.8 ng/L), insulin concentrations during LgHGC were increased (760.8 +/- 350.7 ng/L; P = 0.0022), glucagon concentrations decreased (0.66 +/- 0.4 pmol/L during HGC vs 0.5 +/- 0.4 pmol/L during LgHGC; P = 0.0089), and there was a trend toward an increased total glucose infused (median [range] = 1.61 (1.11-2.54) g/kg and 2.25 (1.64-3.10) g/kg, respectively; P = 0.087). Appetite reduction and decreased body weight (9% +/- 3%; P = 0.006) were observed in all cats. Liraglutide has similar effects and pharmacokinetics profile in cats to those reported in people. With a half-life of approximately 12 h, once daily dosing might be feasible; however, significant effects on appetite and weight loss may necessitate dosage or dosing frequency reductions. Further investigation of liraglutide in diabetic cats and overweight cats is warranted. (C) 2015 Elsevier Inc. All rights reserved.

Phytotoxicity associated to microcystins: a review

Microcystins (MC) are the most studied toxins of cyanobacteria since they are widely distributed and account for several cases of human and animal poisoning, being potent inhibitors of the serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A). The phosphatases PP1 and PP2A are also present in plants, which may also suffer adverse effects due to the inhibition of these enzymes. In aquatic plants, biomass reduction is usually observed after absorption of cyanotoxins, which can bioaccumulate in its tissues. In terrestrial plants, the effects caused by microcystins vary from inhibition to stimulation as the individuals develop from seedling to adult, and include reduction of protein phosphatases 1 and 2A, oxidative stress, decreased photosynthetic activity and even cell apoptosis, as well as bioaccumulation in plant tissues. Thus, the irrigation of crop plants by water contaminated with microcystins is not only an economic problem but becomes a public health issue because of the possibility of food contamination, and this route of exposure requires careful monitoring by the responsible authorities.