Avaliamos importância da NADPH oxidase 2 (NOX2) na produção de espécies reativas de oxigênio (EROs) em ilhotas de ratas alimentadas ou em jejum, incubadas na presença de 2,8 mM ou 16,7 mM de glicose, associada ou não a leucina, com ou sem inibição da NOX2. As ilhotas dos animais alimentados ou em jejum apresentaram reduzida secreção de insulina e altas concentrações de EROs na presença de 2,8 mM de glicose. Esses parâmetros foram invertidos pela adição de inibidores da NOX2. A leucina, que é metabolizada no Ciclo dos Ácidos Tricarboxílicos, também aumentou a secreção de insulina por aumento de ATP, e diminuiu as EROs, devido ao aumento de NADPH, um substrato do sistema antioxidante. Desse modo, quando as ilhotas são submetidas ao jejum, a diminuição da atividade secretória é fundamental para impedir que quantidades maiores de hormônio sejam secretadas, podendo levar a uma hipoglicemia. Porém, na presença de alta concentração de glicose, a ativação das defesas antioxidantes da célula b atenua o excesso de EROS, liberando a secreção de insulina. / We sought to evaluate the importance of NADPH oxidase 2 (NOX2) in the production of reactive oxygen species (ROS) in islets from rats fed or fasted, incubated in the presence of glucose 2.8 mM or 16.7 mM, with or without leucine or inhibition of NOX2. Islets of fed or fasted animals showed reduced insulin secretion and high concentration of ROS in the presence of 2.8 mM glucose. These parameters were reversed by addition of inhibitors NOX2. Leucine that is metabolized in the cycle of Tricarboxylic Acids (TCA) also increased insulin secretion, by increasing ATP, and ROS decreased due to the increase of NADPH, a substrate of the antioxidant system. Thus, when the islets are subjected to fasting, decreased secretory activity is essential to prevent amounts of the hormone be secreted and may lead to hypoglycaemia. However, in the presence of high glucose levels, activation of antioxidant defenses of b cell attenuates the excess of ROS, releasing insulin secretion.

Avaliamos importância da NADPH oxidase 2 (NOX2) na produção de espécies reativas de oxigênio (EROs) em ilhotas de ratas alimentadas ou em jejum, incubadas na presença de 2,8 mM ou 16,7 mM de glicose, associada ou não a leucina, com ou sem inibição da NOX2. As ilhotas dos animais alimentados ou em jejum apresentaram reduzida secreção de insulina e altas concentrações de EROs na presença de 2,8 mM de glicose. Esses parâmetros foram invertidos pela adição de inibidores da NOX2. A leucina, que é metabolizada no Ciclo dos Ácidos Tricarboxílicos, também aumentou a secreção de insulina por aumento de ATP, e diminuiu as EROs, devido ao aumento de NADPH, um substrato do sistema antioxidante. Desse modo, quando as ilhotas são submetidas ao jejum, a diminuição da atividade secretória é fundamental para impedir que quantidades maiores de hormônio sejam secretadas, podendo levar a uma hipoglicemia. Porém, na presença de alta concentração de glicose, a ativação das defesas antioxidantes da célula b atenua o excesso de EROS, liberando a secreção de insulina. / We sought to evaluate the importance of NADPH oxidase 2 (NOX2) in the production of reactive oxygen species (ROS) in islets from rats fed or fasted, incubated in the presence of glucose 2.8 mM or 16.7 mM, with or without leucine or inhibition of NOX2. Islets of fed or fasted animals showed reduced insulin secretion and high concentration of ROS in the presence of 2.8 mM glucose. These parameters were reversed by addition of inhibitors NOX2. Leucine that is metabolized in the cycle of Tricarboxylic Acids (TCA) also increased insulin secretion, by increasing ATP, and ROS decreased due to the increase of NADPH, a substrate of the antioxidant system. Thus, when the islets are subjected to fasting, decreased secretory activity is essential to prevent amounts of the hormone be secreted and may lead to hypoglycaemia. However, in the presence of high glucose levels, activation of antioxidant defenses of b cell attenuates the excess of ROS, releasing insulin secretion.

α-Lipoic acid (LA) has been shown to improve the diabetic cardiac symptoms. However, the underlying mechanisms have not been elucidated precisely. We have reported recently that LA potentially protected neurons from substance-induced apoptosis. We hypothesized that LA could attenuate cardiac cells death induced by oxidative stress derived from high glucose. To test this possibility, we examined the effects of LA on . d-glucose/glucose oxidase (DG/GO, 30. mM/5. mU)-induced injury in rat cardiomyoblast H9c2 cells. We observed that LA pretreatment significantly increased cell viability in DG/GO-challenged cells. LA pretreatment also attenuated DG/GO-induced apoptosis as evidenced by decreases in both nuclear condensation and loss of mitochondrial potential. In addition, LA activated ERK1/2 and moderately increased ROS production. Blockade of ERK1/2 activation by PD98059 completely abolished LA-induced protection against DG/GO challenge. Inhibition of ROS by . N-acetylcysteine abrogated LA-induced ERK1/2 activation and cytoprotection. Furthermore, we observed that the ROS production induced by LA was significantly slower and milder than that by DG/GO. Our results suggest that pretreatment with LA moderately increased ROS production to induce a preconditioning-like effect by ERK1/2 activation thereby increased tolerance of H9c2 cells to DG/GO challenge.

Introduction Oral delivery of proteins faces numerous challenges due to their enzymatic susceptibility and instability in the gastrointestinal tract. In recent years, the polyelectrolyte complexes have been explored for their ability to complex protein and protect them against chemical and enzymatic degradation. However, most of the conventional binary polyelectrolyte complexes (PECs) are formed by polycations which are associated with toxicity and non-specific bio-interactions. The aim of this thesis was to prepare a series of ternary polyelectrolyte complexes (APECs) by introduction of a polyanion in the binary complexes to alleviate the aforementioned limitations. Method Eight non-insulin loaded ternary complexes (NIL APECs) were spontaneously formed upon mixing a polycation [polyallylamine (PAH), palmitoyl grafted-PAH (Pa2.5), dimethylamino-1-naphthalenesulfonyl grafted-PAH (Da10) or quaternised palmitoyl-PAH (QPa2.5)] with a polyanion [dextran sulphate (DS) or polyacrylic acid (PAA)] at 2:1 ratio, in the presence of ZnSO4 (4μM). A model protein i.e., insulin was added to a polycation, prior to addition of a polyanion and ZnSO4 to form eight insulin loaded (IL) APECs. PECs were used as a control to compare APECs. The complexes were characterised by dynamic light scattering (DLS) and transmission electron microscope (TEM). In vitro stability of the complexes was investigated at pH (1.2-7.4), temperature (25˚C, 37˚C and 45˚C) and ionic strength (NaCl-68mM, 103mM and 145mM). Insulin complexation efficiency was assessed by using bovine insulin ELISA assay kit. The in vitro cytotoxicity was investigated on CaCo2 and J774 cells by MTT (3-4,5 dimethyl thialzol2,5 diphenyl tetrazolium bromide) assay. All complexes were evaluated for their haemocompatibility by using haemolysis assay, oxidative stress by reactive oxygen species (ROS) assay and immunotoxicity by in vitro and in vivo cytokine generation assay. The potential of the uptake of complexes across CaCo2 cells was determined by flow cytometry and fluorescent microscopy. The underlying mechanism of transport of complexes was determined by TEER measurement, assessment of FITC-Dextran and insulin transport across CaCo2 cells. 15 Results NIL QPa2.5 APECs (except IL QPa2.5-DS) exhibited larger hydrodynamic sizes (228-468nm) than all other APECs, due to the presence of bulky quaternary ammonium moieties. QPa2.5 APECs exhibited lower insulin association efficiency (≤40%) than other APECs (≥55%) due to a competition between the polyanion and insulin for QPa2.5 leading to reduced association of insulin in the complexes. DS based APECs generally offered higher insulin association efficiency (≥75%) than PAA based APECs (≤55%) due to higher molecular weight (6-10kDa) of DS. In comparison to other complexes, Pa2.5 PECs and APECs were more stable at varying temperature, ionic strength and pH due to the presence of long palmitoyl alkyl chain (C16) which reduced the chain flexibility and provided stronger hydrophobic association. The cytotoxicity of polycations on CaCo2 and J774 cells is rated as PAH>Da10=Pa2.5>QPa2.5. The introduction of PAA in Pa2.5 and Da10 brought most significant improvement in IC50 i.e., 14 fold and 16 fold respectively on CaCo2 cells; 9.3 fold and 3.73 fold respectively on J774 cells. In comparison to other complexes, Da10 (8mgml-1) induced higher haemolytic activity (~37%) due to a higher hydrophobic load of 10 percent mole grafting of dansyl pendants. The entire range of APECs displayed ≤12% ROS generation by the CaCo2 cells. The degree of in vitro TNFα production (QPa2.5≥Da10≥Pa2.5=PAH) and in vitro IL-6 generation (QPa2.5≥Pa2.5=PAH≥Da10) by J774 cells established an inverse relationship of cytotoxicity with the cytokine generation. Similar to MTT data, the introduction of PAA in APECs brought more significant reduction in in vitro cytokine secretion than DS based APECs. Pa2.5-PAA brought the most significant reduction in both in vitro and in vivo cytokine generation. All the formulations were able to significantly reduce original TEER, however did not demonstrate appreciable paracellular permeation of a hydrophilic macromolecular tracer of paracellular transport i.e., FITC Dextran. The uptake study revealed internalisation of APECs predominantly by a transcellular route. Transcellular uptake of IL QPa2.5 (≤73%), IL QPa2.5-DS (67%) was higher than their NIL counterparts, whereas the uptake of NIL Pa2.5 (≤89%), NIL Pa2.5-PAA (42%) was higher than their IL counterparts. Conclusion In essence, amphiphilic APECs have shown polyanion dependent ability to reduce polycation associated toxicity and they are able to facilitate transcellular uptake of insulin across CaCo2 cells.