Class 1A phosphoinositide 3-kinase (PI3K) is essential for beta-cell growth and survival. Although PI3K has been studied extensively in diabetes the effect of alternatively spliced isoforms of the catalytic subunit p85α on beta cell proliferation and survival remains to be defined.We examined expression and signaling of alternatively spliced PI3K regulatory subunits p85α, p55α and p50α in insulinoma cells (INS-1E), an insulin-producing beta cell line. PI3K regulatory isoforms were knocked down by siRNA transfection or overexpressed by adenoviral gene delivery.Knockdown of p85α elevated PI3K activation determined by Akt phosphorylation at baseline and after stimulation with growth factors. In contrast, Akt phosphorylation was inhibited by overexpression of all isoforms of p85α. Correspondingly, p55α and p85α overexpression decreased downstream kinase GSK-3 phosphorylation as well, whereas p50α overexpression resulted in an activation of GSK-3. Moreover, overexpression of p50α and p85α lead to retinoblastoma protein hyperphosphorylation and S-phase entry. Upon challenge of INS-1E cells with a cytotoxic cytokine cocktail, levels of p85α were reduced and p50α was upregulated. Selective overexpression of p50α prevented cytokine induced apoptosis in INS-1E cells.In conclusion, signalling of p50α, p55α and p85α is similar at the level of Akt, but differentially influence downstream GSK-3 activation and cell cycle entry. PI3K isoform p50α induction by cytokines provides a link between regeneration and cell survival under cytotoxic stress in insulin-producing pancreatic beta-cells.