Anchoring of protein kinase A facilitates hormone-mediated insulin secretion

Abstract

Impaired insulin secretion is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). One possible therapeutic agent for NIDDM is the insulinotropic hormone glucagon-like peptide 1 (GLP-1). GLP-1 stimulates insulin secretion through several mechanisms including activation of protein kinase A (PKA). We now demonstrate that the subcellular targeting of PKA through association with A-kinase-anchoring proteins (AKAPs) facilitates GLP-1-mediated insulin secretion. Disruption of PKA anchoring by the introduction of anchoring inhibitor peptides or expression of soluble AKAP fragments blocks GLP-1 action in primary islets and cAMP-responsive insulin secretion in clonal beta cells (RINm5F). Displacement of PKA also prevented cAMPmediated elevation of intracellular calcium suggesting that localized PKA phosphorylation events augment calcium f lux.Subcellular targeting of protein kinases and phosphatases is an efficient mechanism to selectively regulate cellular events by ensuring that phosphorylation only occurs at precise intracellular sites (1-3). Accumulating evidence suggests that the subcellular location of the cAMP-dependent protein kinase (PKA) is a determinant in the preferential phosphorylation of certain PKA substrates (4, 5). Compartmentalization of the PKA holoenzyme is conferred by the association of the regulatory subunit dimer (RII) with a family of A-kinaseanchoring proteins (AKAPs) (6, 7). Anchoring not only places the kinase close to preferred substrates but also positions the PKA holoenzyme at sites where it can optimally respond to fluctuations in the second messenger cAMP (1-3). This higher order of spatial organization may also coordinate effectormediated events that integrate second messenger signals (8-11). For example, insulin secretion from pancreatic beta cells requires the coordinate action of metabolites, hormones, and neurotransmitters (12, 13). A recently identified hormone, glucagon-like peptide 1 (GLP-1), potentiates glucosemediated insulin secretion through activation of PKA (14-16).In this report we demonstrate that the correct subcellular location of PKA is a determinant in the cAMP signaling pathway in response to GLP-1 to induce insulin secretion from pancreatic islets and related cell lines. METHODRII Overlay. The presence of AKAPs in primary rat pancreas was detected by a solid phase RII overlay as previously described (17, 18). Fifty micrograms of total pancreas protein was separated by electrophoresis on an SDS͞10% polyacrylamide gel and electrotransferred to nitrocellulose. Filters were pretreated with either 3 M Ht31 peptide or 3 M Ht31P peptide and then incubated with 32 P-labeled RII␣ overnight at room temperature. RII binding proteins were detected by autoradiography.Preparation of Primary Islets and Transfected RINm5F Cells. Normal rat pancreas was isolated and infused with Hanks' buffer before dissection. Pancreatic islets were isolated by collagenase digestion and plated on Falcon tissue culture dishes. Islets were maintained in culture for up to 5 day...

Important Role of Phosphodiesterase 3B for the Stimulatory Action of cAMP on Pancreatic β-Cell Exocytosis and Release of Insulin

“…However, such a role of cAMP for nutrient-induced insulin release has also been questioned, because inhibition of PKA activity by cAMP analogues (8) or pseudosubstrate for PKA (42) does not inhibit glucose-induced insulin secretion, although the same tools efficiently block secretion in response to adenylyl cyclase activators. It has also been shown that subcellular targeting of PKA by association with anchoring proteins is required for hormone-mediated insulin release but not for glucose-mediated secretion of insulin (43). In contrast to these findings, our results obtained by overexpression of PDE3B, thus lowering the cAMP concentration in ␤-cells, demonstrate a clear detrimental effect on glucose-stimulated insulin secretion.…”

Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells.

“…The lower amount of stimulation observed in islets compared with INS-1 cells may be explained by an increased basal stimulation mediated by glucagon, secreted from ␣-cells in the rat islets. cAMP exerts its effect as a potentiator of nutrient-induced insulin release by the activation of protein kinase A, resulting in phosphorylation of several cellular proteins (19,22). Recently, Xie et al (23) described several binding sites for transcriptional factors in the 5Ј-flanking region of the IA-2 gene, including cAMP response element (CRE), E2F, HSF, YY1, and Sp1.…”

Oscillatory control of insulin secretion

“…The effects of cAMP on the exocytosis machinery involves both PKA-dependent and -independent mechanisms, the latter most likely mediated by Epac (Renström et al, 1997;Seino and Shibasaki, 2005). Many exocytosis-related proteins have been identified as substrates for PKA (Seino and Shibasaki, 2005), and the subcellular targeting of PKA to its effectors via A-kinase anchoring proteins has been found critical for the stimulatory effect of cAMP-elevating agents on insulin secretion (Lester et al, 1997;Fraser et al, 1998;Lester et al, 2001). Capacitance recordings have indicated that PKA mediates the slower cAMP-dependent mobilization of insulin granules, while Epac accounts for the rapid cAMP-dependent potentiation of exocytosis in β-cells (Renström et al, 1997;Eliasson et al, 2003).…”