h-89 and glucagon-like-peptide-1-(7-36)amide

Glucose can activate the mitogen-activated kinases, Erk-1/2, and the ribosomal-S6 kinase, p70(S6K), in beta-cells, contributing to an increase in mitogenesis. However, the signaling mechanism by which glucose induces Erk-1/2 and p70(S6K) phosphorylation activation is undefined. Increased glucose metabolism increases [Ca(2+)](i) and [cAMP], and it was investigated if these secondary signals were linked to glucose-induced Erk-1/2 and p70(S6K) activation in pancreatic beta-cells. Blocking Ca(2+) influx with verapamil, or inhibiting protein kinase A (PKA) with H89, prevented glucose-induced Erk-1/2 phosphorylation. Increasing cAMP levels by GLP-1 potentiated glucose-induced Erk-1/2 phosphorylation via PKA activation. Elevation of [Ca(2+)](i) by glyburide potentiated Erk-1/2 phosphorylation, which was also inhibited by H89, suggesting increased [Ca(2+)](i) preceded PKA for glucose-induced Erk-1/2 activation. Adenoviral-mediated expression of dominant negative Ras in INS-1 cells decreased IGF-1-induced Erk-1/2 phosphorylation but had no effect on that by glucose. Collectively, our study indicates that a glucose-induced rise in [Ca(2+)](i) leads to cAMP-induced activation of PKA that acts downstream of Ras and upstream of the MAP/Erk kinase, MEK, to mediate Erk-1/2 phosphorylation via phosphorylation activation of Raf-1. In contrast, glucose-induced p70(S6K) activation, in the same beta-cells, was mediated by a distinct signaling pathway independent of Ca(2+)/cAMP, most likely via mTOR-kinase acting as an "ATP-sensor."

Topics: Animals; Calcium; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Glyburide; Humans; Islets of Langerhans; Isoquinolines; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Peptide Fragments; Phosphorylation; Proto-Oncogene Proteins c-raf; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Sulfonamides; Verapamil

Whereas the loss of ATP-sensitive K(+) channel (K(ATP) channel) activity in human pancreatic beta-cells causes severe hypoglycemia in certain forms of hyperinsulinemic hypoglycemia, similar channel loss in sulfonylurea receptor-1 (SUR1) and Kir6.2 null mice yields a milder phenotype that is characterized by normoglycemia, unless the animals are stressed. While investigating potential compensatory mechanisms, we found that incretins, specifically glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), can increase the cAMP content of Sur1KO islets but do not potentiate glucose-stimulated insulin release. This impairment is secondary to a restriction in the ability of Sur1KO beta-cells to sense cAMP correctly. Potentiation does not appear to require cAMP-activated protein kinase (PKA) because H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) and KT5720, inhibitors of PKA, do not affect stimulation by GLP-1, GIP, or exendin-4 in wild-type islets, although they block phosphorylation of cAMP-response element-binding protein. The impaired incretin response in Sur1KO islets is specific; the stimulation of insulin release by other modulators, including mastoparan and activators of protein kinase C, is conserved. The results suggest that the defect responsible for the loss of cAMP-induced potentiation of insulin secretion is PKA independent. We hypothesize that a reduced release of insulin in response to incretins may contribute to the unexpected normoglycemic phenotype of Sur1KO mice versus the pronounced hypoglycemia seen in neonates with loss of K(ATP) channel activity.

Topics: Animals; ATP-Binding Cassette Transporters; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Indoles; Insulin; Insulin Secretion; Islets of Langerhans; Isoquinolines; Mice; Mice, Knockout; Peptide Fragments; Potassium Channels; Potassium Channels, Inwardly Rectifying; Protein Kinase C; Pyrroles; Receptors, Drug; Reference Values; Sulfonamides; Sulfonylurea Receptors