Glucagon-Like Peptide-1 Causes Pancreatic Duodenal Homeobox-1 Protein Translocation from the Cytoplasm to the Nucleus of Pancreatic β-Cells by a Cyclic Adenosine Monophosphate/Protein Kinase A-Dependent Mechanism

Introduction: The progression to hyperglycaemia in type 2 diabetes is marked by β-cell insulin secretory dysfunction and cell loss. We have previously demonstrated that apolipoprotein (apo) A-I, the major protein constituent of high density lipoproteins (HDL) increases insulin expression and secretion from β-cells. Clinical data also suggests that pharmacological elevation of HDL levels is associated with improved glycemic control in patients with type 2 diabetes. With the current interest in HDL raising therapeutics, defining the mechanism by which apoA-I acts on insulin secretion is of importance. Objective: To elucidate the cell signalling events responsible for increasing insulin secretion from pancreatic β-cells treated with lipid-free apoA-I. Methods: Ins-1E (rat insulinoma) cells were pre-treated for 30 min with the Protein kinase A (PKA) specific inhibitor H89 (20 μM), soluble and transmembrane adenyl cyclase specific inhibitors (KH7, 30 μM and 2’5’ dideoxyadenosine, 50 μM, respectively) or vehicle control, then incubated for 1 h with lipid-free apoA-I (final concentration 1 mg/mL) under both basal (2.8 mM) and high (25 mM) glucose conditions. The insulin concentration in the culture supernatants was determined by radioimmunoassay and the cells were either lysed for protein analysis by western blotting or treated with 0.1 M HCl for determining cAMP by enzyme immunoassay. Results: Incubation of Ins-1E cells with apoA-I increased insulin secretion up to 3-fold. This increase was no longer apparent when the cells were pre-treated with H89. Incubation with apoA-I increased cAMP accumulation in Ins-1E cells 2.5-fold. This increase was totally inhibited when the cells were pre-incubated with 2’5’ dideoxyadenosine but not by KH7, indicating that transmembrane adenyl cyclase(s) are responsible for this response. ApoA-I also activated the small GTPase Cdc42, which may link cell surface apoA-I receptors with transmembrane adenyl cyclases. Conclusion: ApoA-I increases insulin secretion from pancreatic β-cells via a PKA-dependent mechanism involving transmembrane, but not soluble, adenyl cyclases and possibly Cdc42. This provides a possible explanation of the clinical observations that increased HDL may be beneficial in type 2 diabetes.

Download Full-text

Ca2+-induced Ca2+ Release via Inositol 1,4,5-trisphosphate Receptors Is Amplified by Protein Kinase A and Triggers Exocytosis in Pancreatic β-Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m407673200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45455-45461 ◽

Cited By ~ 72

Author(s):

Oleg Dyachok ◽

Erik Gylfe

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Ryanodine Receptors ◽

Nucleotide Exchange ◽

High Concentration ◽

Β Cells ◽

Pancreatic Β Cells ◽

Inositol 1,4,5 Trisphosphate ◽

Glucagon Like Peptide 1 ◽

Kinase A

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca2+-induced Ca2+release (CICR) in pancreatic β-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca2+spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca2+concentration ([Ca2+]i). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca2+spiking at low or even in the absence of depolarization-dependent elevation of [Ca2+]i. The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca2+, failed to mobilize intracellular Ca2+. On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP3Rs). Therefore, the cell-permeable IP3R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic β-cells were followed by [Ca2+]ispikes in neighboring human erythroleukemia cells, used to report secretory events in the β-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP3Rs is part of the mechanism by which cAMP amplifies insulin release.

Download Full-text