Untersuchung zur Funktion der regulatorischen Untereinheiten p50α, p55α und p85α der Phosphoinositid-3-Kinase in der beta-Zell-Linie INS-1E

Lipid kinase phosphoinositide 3-kinase (PI3K) regulates Akt (protein kinase B, PKB) signalling, which is is pivotal for pancreatic beta-cell growth and survival. PI3K is active as a heterodimer of regulatory and catalytic subunits and inhibited by an excess of regulatory subunits. We examined expression and signalling of PI3K regulatory subunits p85α, p55α and p50α in insulinoma cells (INS-1E) stimulated by cAMP and IGF-1, two potent beta-cell growth factors. PI3K regulatory isoforms p85α, p55α and p50α were knocked down by siRNA transfection or overexpressed by adenoviral gene delivery in INS-1E cells stimulated with glucose, cAMP and IGF-1. PI3K assays were performed using phosphoinositide as a substrate. Activation of Akt, GSK-3, p70S6K, Rb and p44/p42 MAPK were determined by immunoblotting with specific antibodies. Cell cycle regulation was determined by FACS. Regulatory isoforms p85α, p55α and p50α are expressed together with p110 catalytic units in INS-1E cells. Knockdown of p85α elevated PI3K activation, which was inhibited by elevation of p85α and p50α but not p55α. Conversely, a two-fold elevation of all regulatory subunits of PI3K levels by adenoviral gene transfer inhibited Akt phosphorylation.. Whereas overexpression of p55α reduced levels of phosphorylated und non-phosphorylated GSK-3, it did not increase phosphorylation of Rb and p44/p42 MAPK in contrast to overexpressed p50α and p85α. Overexpression of p50α and p85α led to a massive increase in S-phase in contrast to p55α. In insulin-secreting cells, p85α, p50α and p55α behave similarly as in insulin-sensitive tissues by inhibiting Akt. However, downstream signalling of p55α is different from that of p85α and p50α and also does not influence cell cycle regulation or prevent cytotoxic cell death. Thus, PI3K isoforms signal differently downstream of Akt. PI3K isoform p50α and to a lesser extend p85α but not p55α provide a link between proliferation and apoptosis in insulin-producing pancreatic beta-cells.