Apigetrin ameliorates streptozotocin-induced pancreatic ß-cell damages via attenuating endoplasmic reticulum stress.

The pathogenesis of diabetes is associated with dysfunction of pancreatic ß-cells. To ameliorate the ß-cell dysfunction, it has propelled great interest to search pharmacological agents from natural plants. This study explored the protective effect of apigetrin, a flavonoid present in natural plants, against streptozotocin (STZ)-induced cell damages in RINm5F cells and the potential mechanisms. Apigetrin was found to inhibit the elevation of intracellular reactive oxygen species levels, restore the impairment of antioxidant enzymes, and recover the disruption of redox homeostasis in the STZ-treated pancreatic ß-cells. Moreover, treatment of apigetrin significantly suppressed the STZ-induced apoptosis in the analysis of apoptotic sub-G1 population and the protein expressions of cleaved poly(ADP-ribose) polymerase and caspase-3. Furthermore, apigetrin attenuated STZ-induced endoplasmic reticulum (ER) stress, indicated by the reduction of ER stress biomarkers, including overloading of mitochondrial calcium, increase in glucose-regulated protein 78, phosphorylation of protein kinase RNA-like ER kinase and its downstream eukaryotic initiation factor 2α, cleavage of activating transcription factor 6 and caspase-12, up-regulation of CCAAT/enhancer binding protein homologous protein, and induction of spliced X-box binding protein 1. Additionally, pretreatment with 4-phenylbutyric acid, a classic ER stress inhibitor, augmented these beneficial effects of apigetrin. In conclusion, these results demonstrated that apigetrin could improve the STZ-induced pancreatic ß-cell damages via mitigation of oxidative stress and ER stress and supported the application of apigetrin to developing the novel therapeutics of diabetes.

Oligomannuronate prevents mitochondrial dysfunction induced by IAPP in RINm5F islet cells by inhibition of JNK activation and cell apoptosis.

BACKGROUND: Oligomannuronates (OM) are natural products from alginate that is frequently used as food supplement. The aim of this study was to investigate the in vitro protective effects of OM on RINm5F cells against human Islet amyloid polypeptide (IAPP) induced mitochondrial dysfunction, as well as the underlying mechanisms. METHODS: In the present study, we obtained several kinds of OM with different molecular masses, and then we used RINm5F cells as a model to elucidate the involvement of JNK signal pathway in hIAPP-induced mitochondrial dysfunction in pancreatic beta cells, and the protective effects of OM are associated with its ability to attenuate the mitochondrial dysfunction. RESULTS: Our results demonstrated that human IAPP induced mitochondrial dysfunction, as evidence by loss of ΔΨm and ATP content, and decrease in oxygen consumption and complex activities, was accompanied by JNK activation, changes in the expressions of Bcl-2 and Bax proteins, release of cytochrome c (Cyto-c) and apoptosis inducing factor (AIF) from mitochondria into cytosol. Interestingly, the human IAPP induced damage in RINm5F cells were effectively restored by co-treatment of OM. Moreover, JNK activation was required for the OM mediated changes in RINm5F cells. CONCLUSIONS: OM prevented mitochondrial dysfunction induced by human IAPP in RINm5F islet cells through JNK dependent signaling pathways.

Protective effects of γ-aminobutyric acid against H2O2-induced oxidative stress in RIN-m5F pancreatic cells.

BACKGROUND: γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and reported to maintain the redox homeostasis and insulin secretion function of pancreatic ß cells. This study tested the hypothesis that GABA maintains cellular redox status, and modulates glycogen synthase kinase (GSK)-3ß and antioxidant-related nuclear factor erythroid 2-related factor 2 (NRF2) nuclear mass ratio in the H2O2-injured RINm5F cells. METHODS: RINm5F cells were treated with/without GABA (50, 100 and 200 µmol/L) for 48 h and then exposed to 100 µmol/L H2O2 for 30 min. Viable cells were harvested, and dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used to detect reactive oxygen species (ROS) level; cellular redox status and insulin secretion were measured; cell viability was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; mitochondrial membrane potential (MMP) was detected by flow cytometry; relative genes levels were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR); western blotting was used to determine protein expression of GSK-3ß and p-GSK-3ß (Ser9), and nuclear and cytoplasmic NRF2. RESULTS: H2O2 increased ROS production, and induced adverse affects in relation to antioxidant defense systems and insulin secretion. These changes were restored by treatment with 100 and 200 µmol/L GABA. In addition, 100 or 200 µmol/L GABA induced membrane depolarization and increased cell viability. These effects were mediated by Caspase-3, Bcl-2 associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) expression. Western blotting indicated that GABA inhibited GSK-3ß by increasing p-GSK-3ß (Ser9) level, and directed the transcription factor NRF2 to the nucleus. CONCLUSION: In rat insulin-producing RINm5F cells, GABA exerts its protective effect by regulating GSK-3ß and NRF2, which governs redox homeostasis by inhibiting apoptosis and abnormal insulin secretion by exposure to H2O2.

Investigation of Antihyperglycaemic Activity of Banana (Musa sp. Var. Nanjangud rasa bale) Flower in Normal and Diabetic Rats.

BACKGROUND: The vital enzymes of starch digestion and absorption are intestinal α-glucosidases and their inhibition improves postprandial hyperglycaemia, constituting an effective mode of therapy in diabetes. OBJECTIVES: The present study was designed to assess the inhibitory potential of ethanol extract of banana flower (EF) on mammalian α-glucosidases and its pharmacological effects on postprandial hyperglycaemia in normal and alloxan-induced diabetic rats. MATERIALS AND METHODS: EF was evaluated for its inhibitory potential and mode of inhibition on mammalian α-glucosidases. Further, the role of EF and its constituents Umbelliferone (C1) and Lupeol (C2) on glucose uptake using isolated rat hemi-diaphragm and insulinotropic activity using RINm5F (rat insulinoma) cell lines were determined. The phytocomponents in EF were also evaluated using GC-MS. RESULTS: EF illustrated a dose-dependent inhibition for rat intestinal sucrase, maltase and p-nitrophenyl-α-D-glucopyranoside (pNPG) hydrolysis (IC50 values: 18.76±0.22, 25.54±0.10 and 76.42±1.12 µg/ml, respectively) and the mode of inhibition was non-competitive with low Ki values. Oral administration (100-200 mg/kg b.wt.) of EF significantly improved the maltose/glucose-induced postprandial hyperglycaemia in normal and alloxan-induced diabetic rats. EF, C1 and C2 exhibited stimulation of glucose uptake and a dose-dependent glucose-induced insulin secretion at both 4.5 and 16.7 mM glucose concentrations. Further, GC-MS analysis revealed significant levels of steroids (25.61%), diazoprogesterone (21.31%), sesquiterpene (11.78%) and other phytocomponents. CONCLUSION: EF inhibited α-glucosidases besides promoting glucose uptake and insulin secretion, resulting in antihyperglycaemic effect determining EF as a potent anti-diabetic agent.Abbreviations used: mg/dl: milligramsper deciliter, mM: millimolar, b.wt.: body weight.

Antioxidant, antiglycation and insulinotrophic properties of Coccinia grandis (L.) in vitro: Possible role in prevention of diabetic complications.

In an attempt to develop Complementary and Alternative Medicine (CAM) for the treatment of diabetes and related complications, the antidiabetic potential of the mature unripe fruits of Coccinia grandis (CGF) was evaluated. Oxidative stress and glycation plays an important role in manifesting of diabetes and vascular complications. Agents with antioxidant and antiglycation properties may retard these pathological alterations. In this study, the edible plant Coccinia grandis was assessed for in vitro estimation of antioxidant and antiglycation potential and its insulinotrophic properties in RINm5F cells. Antioxidant activity was evaluated as DPPH (1,1-diphenyl-2-picrylhydrazyl), hydrogen peroxide and superoxide anion scavenging activities, whereas the protein glycation inhibitory potential was evaluated using in vitro albumin-fructose glycation model. Glycation inhibition was estimated by different biochemical parameters viz. fructosamine, protein carbonyl group and protein aggregation using thioflavin T fluorescence. C. grandis extract exerted a dose dependent radical scavenging activity and exhibited a significant antiglycation potential. The extract also showed a significant insulinotrophic property with 1.28 and 1.71-fold increase in insulin release when compared to control at 0.25 and 0.50 mg/mL, respectively. These data suggest the possible antidiabetic role of CGF extract, presumably by its antioxidant, antiglycation and insulin secretory effects. Present findings provide experimental evidence that the fruits of C. grandis have potential antidiabetic activity which might be used as a functional food and safe remedy for the treatment of diabetes and associated complications. This study also revealed that the plant can be a promising source for development of natural antiglycating agents and novel insulin secretagogues.

The level of nitric oxide regulates lipocalin-2 expression under inflammatory condition in RINm5F beta-cells.

We previously reported that proinflammatory cytokines (interleukin-1ß and interferon-γ) induced the expression of lipocalin-2 (LCN-2) together with inducible nitric oxide synthase (iNOS) in RINm5F beta-cells. Therefore, we examined the effect of nitric oxide (NO) on LCN-2 expression in cytokines-treated RINm5F beta-cells. Additionally, we observed the effect of LCN-2 on cell viability. First, we found the existence of LCN-2 receptor and the internalization of exogenous recombinant LCN-2 peptide in RINm5F and INS-1 beta-cells. Next, the effects of NO on LCN-2 expression were evaluated. Aminoguanidine, an iNOS inhibitor and iNOS gene silencing significantly inhibited cytokines-induced LCN-2 expression while sodium nitroprusside (SNP), an NO donor potentiated it. Luciferase reporter assay showed that transcription factor NF-κB was not involved in LCN-2 expression. Both LCN-2 mRNA and protein stability assays were conducted. SNP did not affect LCN-2 mRNA stability, however, it significantly reduced LCN-2 protein degradation. The LCN-2 protein degradation was significantly attenuated by MG132, a proteasome inhibitor. Finally, the effect of LCN-2 on cell viability was evaluated. LCN-2 peptide treatment and LCN-2 overexpression significantly reduced cell viability. FACS analysis showed that LCN-2 induced the apoptosis of the cells. Collectively, NO level affects LCN-2 expression via regulation of LCN-2 protein stability under inflammatory condition and LCN-2 may reduce beta-cell viability by promoting apoptosis.

Though Active on RINm5F Insulinoma Cells and Cultured Pancreatic Islets, Recombinant IL-22 Fails to Modulate Cytotoxicity and Disease in a Protocol of Streptozotocin-Induced Experimental Diabetes.

Interleukin (IL)-22 is a cytokine displaying tissue protective and pro-regenerative functions in various preclinical disease models. Anti-bacterial, pro-proliferative, and anti-apoptotic properties mediated by activation of the transcription factor signal transducer and activator of transcription (STAT)-3 are key to biological functions of this IL-10 family member. Herein, we introduce RINm5F insulinoma cells as rat ß-cell line that, under the influence of IL-22, displays activation of STAT3 with induction of its downstream gene targets Socs3, Bcl3, and Reg3b. In addition, IL-22 also activates STAT1 in this cell type. To refine those observations, IL-22 biological activity was evaluated using ex vivo cultivated murine pancreatic islets. In accord with data on RINm5F cells, islet exposure to IL-22 activated STAT3 and upregulation of STAT3-inducible Socs3, Bcl3, and Steap4 was evident under those conditions. As these observations supported the hypothesis that IL-22 may exert protective functions in toxic ß-cell injury, application of IL-22 was investigated in murine multiple-low-dose streptozotocin (STZ)-induced diabetes. For that purpose, recombinant IL-22 was administered thrice either immediately before and at disease onset (at d4, d6, d8) or closely thereafter (at d8, d10, d12). These two IL-22-treatment periods coincide with two early peaks of ß-cell injury detectable in this model. Notably, none of the two IL-22-treatment strategies affected diabetes incidence or blood glucose levels in STZ-treated mice. Moreover, pathological changes in islet morphology analyzed 28 days after disease induction were not ameliorated by IL-22 administration. Taken together, despite being active on rat RINm5F insulinoma cells and murine pancreatic islets, recombinant IL-22 fails to protect pancreatic ß-cells in the tested protocols from toxic effects of STZ and thus is unable to ameliorate disease in the widely used model of STZ-induced diabetes.

Toxicidade de aminoacetona e células produtoras de insulina / Cytotoxity of aminoacetone on insulin-producing cells

Danos induzidos por hiperglicemia em tecidos no diabetes são caracterizados por quatro mecanismos conectados: aumento do fluxo metabólico através da via do poliol, ativação da proteína quinase C (PKC), aumento da atividade da via das hexosaminas e aumento da produção intracelular dos precursores dos produtos finais de glicação avançada (AGEs). Entre eles, os derivados de metilglioxal, um potente agente de modificação de proteínas e DNA, têm sido associados a complicações microvasculares no diabetes: nefropatia, retinopatia e neuropatia. O metilglioxal é produzido a partir das trioses fosfato, acetona e aminoacetona, um catabólito de treonina e glicina, gerado na matriz mitocondrial. A aminoacetona sofre oxidação enzimática, catalisada por aminoxidase sensível a semicarbazida (SSAO), ou química, catalisada por íons de cobre e ferro, produzindo metilglioxal, H2O2 e NH4 +. Sabendo que metilglioxal e H2O2 são capazes de induzir apoptose e/ou necrose em células produtoras de insulina (RINm5f) propomos uma possível atividade pró-oxidante da aminoacetona sobre células beta do pâncreas. O tratamento destas linhagens com aminoacetona/Cu(II) aumentou a morte celular, fluxo de Ca2+ intracelular, produção de NO, fragmentação do DNA, depleção dos níveis de glutationa reduzida (GSH), expressão gênica da proteína apoptótica Bax, enzimas antioxidantes - glutationa peroxidase (GPx), glutationa redutase (GRd), catalase e isoformas de superóxido dismutases (CuZnSOD e MnSOD) - e óxido nítrico sintase induzida (iNOS). Embora as concentrações normais e patológicas da aminoacetona, provavelmente seja muito menores que as usadas nos experimentos, sugerimos que, em tecidos de diabéticos, um acúmulo da aminoacetona em longo prazo pode conduzir a danos oxidativos e eventualmente morte das células beta do pâncreas.

Tissue damages induced by hyperglycemia in diabetics are characterized by four linked mechanisms: increased flux through the polyol pathway, protein kinase C (PKC) activation, increased hexosamine pathway activity and intracellular production of advanced glycation end product (AGE) precursors. The production of AGEs by modifying proteins and DNA agent, such as methylglyoxal, has been implicated in microvascular complications in diabetes: nephropathy, retinopathy and neuropathy. Methylglyoxal is putatively produced in vivo from trioses phosphate, acetone and aminoacetone, a catabolite of threonine and glycine synthesized in the mitochondrial matrix. Aminoacetone has been reported to undergo semicarbazide sensitive amine oxidase- catalyzed and copper- and iron-catalyzed oxidations by molecular oxygen to methylglyoxal, NH4 + ion and H2O2. Considering that methylglyoxal and H2O2 have been found to promote apoptosis/necrosis to insulin-producing cells (RINm5f), we propose a possible pro-oxidant role of aminoacetone in pancreatic beta-cells. Treatment of RINm5f cells with aminoacetone plus Cu(II) ion promotes an increase of non-viable cells, influx of Ca2+ ions, NO production, DNA fragmentation, depletion of reduced glutathione (GSH) levels, and increased mRNA expression of pro-apoptotic protein (Bax), antioxidant enzymes - glutathione peroxidase (GPx), glutathione reductase (GRd), MnSOD, CuZnSOD and catalase - and inducible nitric oxide synthase (iNOS). Although both normal and pathological concentrations of aminoacetone are probably much lower than those used here, it is tempting to propose that excess aminoacetone in diabetic patients, at long term, may drive oxidative damage and eventually death of pancreatic beta-cells.