== (a) Quantitative RT-PCR analysis of the GLP-1 receptor was performed in PIC-transfected MIN6 cells with or without Ex4 (10 nM and 100 nM, n = 3). Fulminant type 1 diabetes mellitus (FT1DM) is a severe subtype of type 1 diabetes characterized by extremely acute and severe insulin insufficiency as a result of almost complete destruction of the pancreatic beta cells even at clinical onset [1]. It is commonly observed in East Asia, where it accounts for approximately 20% of acute-onset type 1 diabetes cases in Japan [2] and 7. 1% of all type 1 diabetes cases in South Korea [3]. It is likely that viral infection contributes to the pathogenesis of FT1DM. A nationwide survey in Japan revealed that 72% of FT1DM cases included a history of flu-like symptoms prior to onset [2]. Anti-enterovirus, anti-human herpesvirus 6, and MDR-1339 anti-cytomegalovirus antibody levels are increased in some FT1DM patients [2]. In the pancreas of patients with FT1DM, enteroviral RNA was directly detected [4]. Recently, it was reported that viral infections may be a possible trigger in beta cell destruction even in type 1A diabetes, which was supposed to account for a major portion of type 1 diabetes cases [5]. Thus, an investigation of the mechanism of beta cell destruction via viral infection is important to clarify the pathophysiology of both FT1DM and type 1A diabetes. Glucagon-like peptide-1 (GLP-1) is an incretin hormone with multiple physiological roles in pancreatic beta cells, including activation of insulin secretion, enhancement of insulin gene transcription and insulin biosynthesis, stimulation of beta cell proliferation, and inhibition of cytokine- [68] and lipotoxicity-induced [9] beta cell apoptosis. We hypothesized that exendin-4 (Ex4), GLP-1 analogue, could also inhibit beta cell apoptosis caused by viral infection. Initially we investigated the mechanism of beta cell destruction in a viral infectious situation and the protective effect of Ex4 by transfecting polyinosinic: polycytidylic acid (PIC) into MIN6 cells, a mouse-derived beta cell line [10]. PIC is a synthetic analogue of viral dsRNA [11], which is known to be a strong inducer of the innate immune responses against viral infection [12] and is often used to mimic viral infection bothin vivoandin vitro[1315]. Furthermore, we extended our study to include insulin-producing cells differentiated from human induced pluripotent stem (iPS) cells to establish a viral infection model of human pancreatic beta cells and to evaluate the anti-apoptotic effect of Ex4 on MDR-1339 human insulin-producing cells. == Materials and Methods == == Cell Culture == MIN6 cells, a mouse-derived MDR-1339 beta cell line [10], were cultured at 37C with 5% CO2in DMEM (SigmaAldrich, St . Louis, MO, USA) containing 450 mg/dl glucose supplemented with 10% FBS (SigmaAldrich), 100 MDR-1339 U/ml penicillin (Nacalai Rabbit polyclonal to AKR1C3 Tesque, Kyoto, Japan), 100 g/ml streptomycin (Nacalai Tesque), and 100 M 2-mercaptoethanol (Nacalai Tesque). 409B2 cells, a human iPS cell line derived from a healthy individual, were purchased from RIKEN Bioresource Centre Cell Bank (Ibaraki, Japan). 409B2 cells were cultured over the Mitomycin C-treated SNL feeder cells at 37C with 5% CO2in Primate ES medium (ReproCELL, Kanagawa, Japan) supplemented with 4 ng/ml recombinant human basic fibroblast growth factor (Wako, Osaka, Japan) and 500 U/ml penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). At 7080% confluence, 409B2 cells were induced to insulin-producing cells using the differentiation protocol described previously [16]. Briefly, cells were first differentiated into endodermal cells expressing sex-determining region Y-box 17 (stage 1, 4 days), then into pancreatic progenitor cells expressing MDR-1339 pancreatic and duodenal homeobox-1 (stage 2, 6 days), and finally into insulin-producing cells (stage 3, 12 days). The iPS cell study protocol was approved by the Ethics Committee of Osaka University. == dsRNA transfection == The synthetic dsRNAs, PIC, were purchased from SigmaAldrich and used at a concentration of 10 g/ml. Transfection of PIC was performed with Lipofectamine 2000 (Life Technologies). In the control study, only the Lipofectamine reagent was added to the medium. After MIN6 cells were cultured for 24 h in 12-well plates at a density of 5 105cells/well for the gene expression analysis and 1 106cells/well for the caspase-3 activity assay, cells were transfected with PIC according to the manufacturers instructions. Five hours later the medium was replaced with DMEM containing 10% FBS and cultured for 19 h prior to evaluation. Meant for the immunocytochemistry analysis, MIN6 cells were cultured in an 8-well holding chamber slide (Thermo Scientific, Chi town, IL, USA) at a density of 2. 5 105cells/well and.