Among the downstream targets of Akt are the Ser/Thr glycogen synthase kinase3�� (GSK3��), and the members Nutlin-3a structure of the Forkhead BoxO (FoxO) family of transcription factors [41], [42]. Akt mediates cell survival through the phosphorylation of GSK3�� which has been proposed as a promising target for beta-cell protection [43]. The phosphorylation of GSK3�� by Akt (inhibits kinase activity) positively affects beta-cell mass and function while its dephosphorylation (kinase activation) enhances beta-cell death [44], [45]. The transcription factors FoxO consist of three members; FoxO1, FoxO3A, and FoxO4, which are all inactivated by Akt [46]. In pancreatic beta-cells, FoxO1 is predominantly expressed while FoxO3A is expressed at a lower level.

Activation of Akt signaling mediates the phosphorylation of the FoxO factors which leads to the nuclear exclusion and then inhibition of the FoxO transcriptional program [47], [48]. In pancreatic beta-cells, it has been shown that FoxO3A specifically controls basal expression of IRS2; this participates to the maintenance of a normal beta-cell mass and function [49]. We have recently shown that JNK3, in contrast to JNK1 and JNK2, has a protective effect in pancreatic beta-cells [12]. We here propose that JNK3 mediates at least partly its protective effect against cytokines through functional preservation of the anti-apoptotic IRS2/Akt2 signaling pathway. Methods Cell Culture The INS-1E cell line [50] was grown in RPMI-1640 medium (Invitrogen, Basel, Switzerland) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) supplemented with 1 mmol/l sodium pyruvate, 10 mmol/l HEPES (pH 7.

6), and 50 ��mol/l ��-mercaptoethanol. Cells were incubated in a humidified atmosphere of 5% CO2 at 37��C. Cell Transfection and Treatments Cells were incubated overnight at a density of 0.6��106 in six-well plates with antibiotic free medium. Small interfering RNA (siRNA) duplexes targeting Jnk1, Jnk2, Jnk3, or the green fluorescent protein (GFP) were mixed with LipofectamineTM2000 reagent according to the manufacturer��s instructions (Invitrogen, Basel, Switzerland). siRNA-Lipofectamine complexes were added to the cells and incubated for 2 days. Cells were then treated with a cocktail of cytokines: recombinant rat IL-1�� (10 ng/ml, R&D systems, Minneapolis, MN, USA), TNF-�� (10 ng/ml, Sigma-Aldrich, Switzerland), and IFN�� (100 ng/ml, Sigma-Aldrich, Switzerland) at the indicated times (see legend figures). For experiments aimed at characterizing insulin-signaling, cells were starved (serum-free) overnight in media supplemented with 2 mmol/l glucose. Cells were then stimulated with recombinant human insulin (100 nmol/l, Sigma-Aldrich, Switzerland) for 30 minutes and processed for protein extract Anacetrapib preparations.