Inhibitory effect of IGF-I on type 2 nitric oxide synthase expression in Ins-1 cells and protection against activation-dependent apoptosis - Involvement of phosphatidylinositol 3-kinase

Challenge of Ins-1 cells, a rat beta-pancreatic cell line, with lipopolysac charide (LPS) and interferon-gamma (IFN-gamma) promoted the expression of t ype 2 nitric oxide synthase (NOS-2) in a cooperative way. Treatment of Ins- 1 cells with IGF-I significantly inhibited the expression of NOS-2, especia lly at subsaturating concentrations of LPS and IFN-gamma. The inhibitory ef fect of IGF-I on NOS-2 expression was abrogated when cells were incubated w ith wortmannin or LY294002, two inhibitors of phosphatidylinositol 3-kinase . Transient expression of the p110 subunit of phosphatidylinositol 3-kinase impaired the LPS and IFN-gamma-dependent NOS-2 promoter activity in cells transfected with a 1-kb fragment corresponding to the 5'-flanking region of the NOS-2 gene. However, expression of a dominant negative form of p85 abo lished the inhibitory action of IGF-I on the NOS-2 promoter activity. Analy sis of the decreased NOS-2 promoter activity in cells incubated with IGF-I showed a lower nuclear factor kappa B binding as determined by electrophore tic mobility shift assays. The synthesis of NO, produced after LPS and IFN- gamma challenge, triggered an apoptotic response in these cells. IGF-I redu ced apoptosis mainly through the decreased synthesis of NO. However, in act ivated cells treated with N-[3-(aminomethyl)benzyl]acetamidine, a specific NOS-2 inhibitor, IGF-I completely abolished the NO-independent apoptosis. T his protection from apoptosis was dependent on phosphatidylinositol 3-kinas e activity. These results suggest an important anti-inflammatory and anti-a poptotic role for IGF-I in beta-pancreatic cells, with both actions dependi ng on the activation of phosphatidylinositol 3-kinase.