Glucose-dependent insulinotropic polypeptide (GIP) acts as a glucose-dependent growth factor for -cells. Here we show that GIP and glucose also act synergistically as anti-apoptotic factors for -cells, using the welldifferentiated -cell line, INS-1. Mitogenic and anti-apoptotic signaling of GIP were dependent upon pleiotropic activation of protein kinase A (PKA)/cAMP regulatory element binder (CREB), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase)/PKB signaling modules. The signaling modules activated by GIP were dependent on glucose metabolism and calcium influx and were tightly linked by multiple activating and inhibiting cross-talk. These interactions included: (i) a central role of tyrosine phosphorylation for stimulation of PKA/CREB, MAPK and PI3-kinase/PKB, (ii) inhibition of PKA/CREB by the MAPK pathway at the level of MAPK kinase-1 or downstream, (iii) activation of MAPK signaling by PI3-kinase and PKA at the level of extracellular-signal regulated kinase 1/2 or upstream, and (iv) activation of PKB by MAPK and PKA signaling at the level of PKB or upstream. Furthermore, we demonstrated inhibition of CREB signaling by Ca 2+ / calmodulin kinase I/IV. These results indicated that GIP acts as a mitogenic and anti-apoptotic factor for -cells by pleiotropic activation of tightly linked signaling pathways in -cells.

The p85␣ regulatory subunit of class I A phosphoinositide 3-kinases (PI3K) is derived from the Pik3r1 gene, which also yields alternatively spliced variants p50␣ and p55␣. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that wild type ES cells express virtually no monomeric p85␣. Although, IGF-1-stimulated PI3K activity associated with insulin receptor substrates was unaltered in all cell lines, p85␣-null ES cells showed diminished protein kinase B activation despite increased PI3K activity associated with the p85␤ subunit. Furthermore, p85␣-null cells demonstrated growth retardation, increased frequency of apoptosis, and altered cell cycle regulation with a G 0 /G 1 cell cycle arrest and up-regulation of p27 KIP , whereas signaling through CREB and MAPK was enhanced. These phenotypes were reversed by re-expression of p85␣ via adenoviral gene transfer. Surprisingly, all ES cell lines could be differentiated into adipocytes. In these differentiated ES cells, however, compensatory p85␤ signaling was lost in p85␣-null cells while increased signaling by CREB and MAPK was still observed. Thus, loss of p85␣ in ES cells induced alterations in IGF-1 signaling and regulation of apoptosis and cell cycle but no defects in differentiation. However, differentiated ES cells partially lost their ability for compensatory signaling at the level of PI3K, which may explain some of the defects observed in mice with homozygous deletion of the Pik3r1 gene.Phosphoinositide 3-kinase (PI3K) 1 generates phosphorylated phosphoinositides (PI), which serve as crucial second messengers for a wide range of biological functions including mitogenesis, survival, differentiation, and cytoskeletal organization. PI3Ks are divided in three major classes according to substrate specificity, amino acid sequence, and homology of their lipid kinase domains. Class IA PI3K phosphorylates phosphatidylinositol (PtdIns), PtdIns 4-phosphate, and PtdIns 4,5-bisphosphate and are active as heterodimers consisting of regulatory and catalytic subunits. Activation of class 1A PI3K occurs by receptor-tyrosine kinases like the insulin-like growth factor-1 (IGF-1) receptor either by direct binding to tyrosine-phosphorylated pYMXM and pYXXM motifs of the IGF-1 receptor ␤-chain and insulin receptor substrates (IRS) (1-4).There are multiple regulatory and catalytic subunits of class 1A PI3K. These regulatory subunits are derived from three different genes and can be classified according to their molecular structure. The full-length regulatory subunits are derived from distinct genes, the Pik3r1 (p85␣) and Pikr2 (p85␤) genes and are comprised of an NH 2 -terminal SH3 domain and a BCR homology region flanked by two proline-rich sites (1-4). In addition, the Pik3r1 gene yields smaller splicing variants of p55kDa (p55␣; also called AS53) and 50 kDa (p50␣) (5-7). Another short regulatory subunit is p55␥; this is stru...

A BSTRACT : Protein kinase B/Akt (PKB/Akt) is activated by phosphatidylinositol 3-kinase (PI 3-K) and is a central mediator of cellular proliferation and protection against apoptosis. Insulin, insulin-like growth factor (IGF-1), and glucagon-like peptide-1 (GLP-1) act as glucose-dependent growth factors for pancreatic ␤ -cells. We assessed signaling pathways and stimulation patterns of PKB/Akt activation by these ligands in the ␤ -cell line INS-1. Insulin, IGF-1, and GLP-1 induced distinctive time dependent, dose dependent, and glucose dependent phosphorylation of PKB/Akt. Insulin and IGF-1 stimulated PI 3-K activity was mainly associated with insulin receptor substrate (IRS) isoforms IRS-1 and IRS-2 and less so with the IRS-isoform Grb -2 associated binder-1 (Gab-1). In contrast, GLP-1 induced PI 3-K activity mainly in Gab-1 and also in IRS-2 immunoprecipitates, although in an attenuated kinetic. Thus, activation pathways of PKB/Akt by insulin, IGF-1, and GLP-1 converge at the level of IRS-isoforms and PI 3-K inducing differential activation of PKB/Akt. These data indicate an essential role of PKB/Akt in regulation of ␤ -cell proliferation.

Activation of the G-protein-coupled receptor for glucose-dependent insulinotropic polypeptide facilitates insulin-release from pancreatic beta-cells. In the present study, we examined whether glucose-dependent insulinotropic polypeptide also acts as a growth factor for the beta-cell line INS-1. Here, we show that glucose-dependent insulinotropic polypeptide induced cellular proliferation synergistically with glucose between 2.5 mM and 15 mM by pleiotropic activation of signaling pathways. Glucose-dependent insulinotropic polypeptide stimulated the signaling modules of PKA/cAMP regulatory element binder, MAPK, and PI3K/protein kinase B in a glucose- and dose-dependent manner. Janus kinase 2 and signal transducer and activators of transcription 5/6 pathways were not stimulated by glucose-dependent insulinotropic polypeptide. Activation of PI3K by glucose-dependent insulinotropic polypeptide and glucose was associated with insulin receptor substrate isoforms insulin receptor substrate-2 and growth factor bound-2 associated binder-1 and PI3K isoforms p85alpha, p110alpha, p110beta, and p110gamma. Downstream of PI3K, glucose-dependent insulinotropic polypeptide-stimulated protein kinase Balpha and protein kinase Bbeta isoforms and phosphorylated glycogen synthase kinase-3, forkhead transcription factor FKHR, and p70S6K. These data indicate that glucose-dependent insulinotropic polypeptide functions synergistically with glucose as a pleiotropic growth factor for insulin-producing beta-cells, which may play a role for metabolic adaptations of insulin-producing cells during type II diabetes.