Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes.

Abstract

Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated ␤-cell destruction. In insulin-producing RINm5F cells, overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined whether overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Zn superoxide dismutase (SOD) can provide protection for bioengineered RINm5F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F control cells to interleukin-1␤ (IL-1␤) alone or a combination of IL-1␤, tumor necrosis factor-␣, and ␥-interferon resulted in a time-and concentration-dependent decrease of cell viability in the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1␤ alone caused only a moderate reduction of viability in the range of 25%, the cytokine mixture induced a significant loss of viability of >75%. This increased toxicity of the cytokine mixture compared with that of IL-1␤ alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic synergism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn SOD protected against toxicity of the cytokine mixture but not against that of IL-1␤ alone. The reduction of cytokine-mediated toxicity was evident also because of an increased proliferation rate and a drastic decrease in the cell death rate. The improved antioxidant defense status did not prevent the activation of iNOS after cytokine exposure. However, RINm5F cells overexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn SOD overexpression was apparently because of an inactivation of ROS generated in the signal cascades of the cytokines. Overexpression of cytoprotective enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.

Increasing uncoupling protein-2 in pancreatic beta cells does not alter glucose-induced insulin secretion but decreases production of reactive oxygen species

“…These data are in agreement with recent investigations demonstrating that high glucose concentrations do not increase but rather decrease ROS generation in rat pancreatic beta cells [42]. Mitochondrial ROS generation has also been reported in beta cells after cytokine exposure [33]. Our results show that increasing UCP2 protein levels promoted a decrease in IL1-β-induced ROS production.…”

“…However, our control cells did not exhibit any increase in ROS generation after short (60 min) or long (24 h) exposure to 20 mmol/l glucose (data not shown). Mitochondrial ROS production in the pancreatic beta cell is also known to increase after administration of cytokines [33]. For this reason, the possibility that increased expression of UCP2 may act on IL1β-induced ROS production was Cells were cultured and oxygen consumption measured as described in the Materials and methods section.…”

Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells

“…The signal pathways of pro-inflammatory cytokines involve binding to specific receptors, activation of mitogen-and stress-activated protein kinases and mobilisation of transcription factors such as NF-κB, STAT-1 and c-myc, which mediate upor down-regulation of gene expression and beta cell apoptosis [4]. In particular the generation of nitric oxide (NO) by induction of inducible nitric oxide synthase (iNOS) and of oxygen free radicals by unknown mechanisms are thought to participate in the signal pathways of programmed cell death and the direct oxidative attack of beta cell structures [3,6,7].…”

IL-1β induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis

“…We hypothesize that the beta cell, when exposed to IL-1β initiates a protective response in competition with a series of deleterious events, and that in beta cells the deleterious events prevail [3]. In support of this, over-expression in islet cells of scavengers of free radicals, such as catalase and glutathione peroxidase, reduces the deleterious effects of cytokines on beta cells [11].…”

“…The presence of the toxic peroxynitrite is a signal to higher production of catalase and glutathione peroxidase to convert peroxinitrite to oxygen and water. Overexpression of catalase, superoxide dismutase and glutathione peroxidase in rat insulinoma (RIN) cells protects against the toxicity of nitric oxide [78] as well as against cytokines [11]. The observed increased expression of catalase (threefold) in the BB-DP islets is apparently insufficient to protect against the IL-1β induced free radicals.…”