The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is a major regulator of postprandial insulin secretion in mammals. Recent studies have suggested that GIP is a potent stimulus for protein kinase activation of the PI3kinase/PKB cascade. We hypothesized that GIP could regulate cell fate and sought to investigate the underlying mechanisms involved in GIP stimulation of cell survival using the β(INS-1) cell line and human pancreatic islets. In response to GIP stimulation, β(INS-1) cell PKB was phosphorylated at the two known phosphosites, threonine (Thr) 308 and serine (Ser) 473, with maximal levels reached after 60 minutes (n = 3, p < .05). GIP-induced phosphorylation of PKB was also shown with human pancreatic islets. The PI3Kinase inhibitor wortmannin completely blocked GIP and forskolin induced PKB Ser 473 phosphorylation. Pharmacologic experiments excluded roles for MAP kinase (Mek)1/2, protein kinase A, Epac, Src, and the EGF receptor in GIP stimulated PKB activation. Phosphorylation of PKB Ser 473 by GIP was ablated by the Ca2+ chelator, EGTA, and the intracellular Ca 2+ release blocker, thapsigargin, indicating a role for calcium signalling. Arachidonic acid (AA) phosphorylated PKB at Ser 473 and GIP stimulated AA release in β(INS-1) cells. The PLA2 inhibitor effectively ablated PKB ser 473 phosphorylation. β(INS-1) cell expression of β-ARKct (β-adrenergic receptor kinase C-terminal tail), a peptide inhibitor of Gβγ, resulted in greatly reduced PKB Ser 473 phosphorylation upon GIP stimulation. GIP promotes β-cell survival under conditions of either high fat coupled with high glucose or complete glucose deprivation. GIP reduced caspase-3 activity in INS-1 cells exposed to high fat and glucose for 24 hours. Studies using both dominant negative and constitutively active forms of PI3kinase and GIP stimulation demonstrated a role for PKB in protecting against glucose deprivation. These data therefore suggest that GIP is able to regulate INS-1 cell survival via Gβγ coupling and dynamic control of PKB phosphorylation via PI3Kinase and intracellular Ca2+ signaling and lend further support to the notion that GIP regulation of PKB signaling is critical for its regulation of cell fate. The impact of GIP on β-cell survival is of great importance for developing treatment against both type 1/2 diabetes. Thus, we hypothesized that GIP can increase survival of INS-1 :158} cells through a PKB/akt-dependent mechanism.
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