Glucagon-Like Peptide 1 Elevates Cytosolic Calcium in Pancreatic  -Cells Independently of Protein Kinase A

Abstract

Glucagon-like peptide 1 (7-36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion, by activation of adenylate cyclase and subsequent elevation of cytosolic free calcium, [Ca2+]cyt. We investigated the role of protein kinase A (PKA) in GLP-1 signal transduction, using isolated mouse islets as well as the differentiated beta-cell line INS-1. Two specific inhibitors of PKA, (Rp)-adenosine cyclic 3',5'-phosporothioate (Rp-cAMPS, up to 3 mM) and KT5720 (up to 10 microM), did not inhibit the GLP-1-induced [Ca2+]cyt elevation. Another PKA inhibitor, H-89, reduced the [Ca2+]cyt elevation only when applied at high concentrations (10-40 microM), higher than sufficient for PKA inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably PKA-independent processes such as glucose-induced [Ca2+]cyt elevations and intracellular calcium storage. This suggests a PKA-independent action of H-89. Similarly to H-89, the potent but unselective protein kinase inhibitor staurosporine inhibited the GLP-1-induced [Ca2+]cyt elevation only at high concentrations, at which it also inhibited glucose-induced [Ca2+]cyt elevations. The same observations as with GLP-1 were made when adenylate cyclase was stimulated with forskolin, for selective examination of signal transduction downstream of receptor and G protein. Our results suggest that the GLP-1-induced [Ca2+]cyt elevation is mediated independently of PKA and thus belongs to the yet-little-characterized ensemble of effects that are mediated by binding of cAMP to other target proteins.

International Union of Pharmacology. XXXV. The Glucagon Receptor Family

“…Distinct amino acids within the amino-terminal domain of the receptor are crucial for ligand binding (Parker et al, 1998;Tibaduiza et al, 2001;Wilmen et al, 1997), and the region encompassing transmembrane domains 1 to 3 is also involved in ligand binding (Xiao et al, 2000b). Different domains in the third intracellular loop of the GLP-1 receptor are responsible for specific G proteincoupling (and G␣ s , G i , and G o activation) (Hallbrink et al, 2001), and in the best studied cellular model, islet ␤-cells, GLP-1 receptor signaling acts predominantly via G s to increase cAMP accumulation; however, activation of downstream signaling pathways may occur in a protein kinase A-independent manner (Bode et al, 1999;Holz et al, 1999). GLP-1-mediated closure of ATP-sensitive potassium (K ATP ) channels, and the differential effect of ADP levels on K ATP channel closure may provide a cellular mechanism for the glucose-sensitivity of GLP-1 action in ␤-cells (Light et al, 2002).…”

“…GLP-1-binding at pancreatic ␤-cells increases free cytosolic calcium concentrations after cell depolarization in some but not all cell types (Goke et al, 1989b;Lu et al, 1993;Yada et al, 1993;Holz et al, 1995Holz et al, , 1999Bode et al, 1999). GLP-1-dependent stimulation of intracellular calcium may occur via a ryanodine-sensitive pathway (Holz et al, 1999), and in a cAMPdependent, protein kinase A-independent manner through small G proteins distinct from G s (Kang et al, 2001;Kashima et al, 2001).…”

Regulation of ERK1 and ERK2 by Glucose and Peptide Hormones in Pancreatic β Cells

“…Glucose metabolism is coupled to Ca 2ϩ influx, which causes Ca 2ϩ release from intracellular stores, through ryanodine and perhaps also IP 3 receptors (44). GLP-1 through cAMP also induces Ca 2ϩ release via Epac2 from intracellular stores (2,59,64). The actions of GLP-1 and glucose converge at this Ca 2ϩ release step, and potentiation of the response may occur as a consequence of the mechanisms by which each agent works on Ca 2ϩ release.…”

Critical Role of cAMP-GEFII·Rim2 Complex in Incretin-potentiated Insulin Secretion

“…It is thought generally that the potentiating effects of incretins on insulin secretion are mediated mainly by the cAMP/PKA signaling pathway in the ␤-cells (3,21,22). However, it has been suggested that incretins also exert their effects in the ␤-cells in a cAMP-independent manner (23)(24)(25). For example, GLP-1 has been suggested to mobilize intracellular calcium by activation of inositol 1,4,5-trisphosphate receptor channels (23).…”

“…For example, GLP-1 has been suggested to mobilize intracellular calcium by activation of inositol 1,4,5-trisphosphate receptor channels (23). GLP-1 has also been shown to elevate [Ca 2ϩ ] i (24) as well as to stimulate insulin gene promoter activity (25) by a PKA-independent mechanism in a ␤-cell-derived cell line.…”