Abstract
Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.
Highlights
Type I or insulin-dependent diabetes mellitus is an autoimmune disease causing specific destruction of the insulin-producing  cells of the islets of Langerhans [1]
This was because these dysfunctions were induced by IFN-␥ alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1 is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse  cells but does not contribute to impaired glucose-stimulated insulin secretion
Stimulation of inducible nitric oxide synthase (iNOS) Expression in CDM3D Cells by IL-1 and IFN-␥—The effect of the pro-inflammatory cytokines on induction of iNOS was tested using the conditionally immortalized murine Tc-Tet cells that had been previously modified to stably express Bcl-2. These cells, referred to as CDM3D, have been shown to be easier to cultivate in standard medium, with a much reduced basal level of apoptosis, to fully conserve the capability of the parental cell line to be growth-arrested by tetracycline, and to secrete insulin with a normal glucose dose dependence [17]
Summary
Type I or insulin-dependent diabetes mellitus is an autoimmune disease causing specific destruction of the insulin-producing  cells of the islets of Langerhans [1]. Important for the present study, it has been recently reported that interference with the intracellular IL-1 signaling pathway can be achieved by overexpressing either the Toll domain of MyD88 or the adaptor containing a Death domain mutation This mutation (MyD88F56N) is similar to the lpr mutation found in the Death domain of the FAS receptor, which blocks the Fas-Fadd interaction [11]. To evaluate the potential of blocking this pathway by gene transfer in  cells and the consequence of this inhibition on NF-B activation, NO production, and GSIS in response to cytokine treatment, we used the conditionally immortalized Tc-Tet cells These cells have been established in culture from islets of transgenic mice expressing the SV-40 T antigen under the control of the tetracycline operator/tetracycline transactivator system [15]. These cells treated with IFN-␥ plus IL-1 or IFN-␥ alone showed reduced GSIS, which could be explained by an IFN-␥-induced decrease of their intracellular insulin content and a decrease in the secretory activity stimulated by glucose
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