Structure–activity relationships of glucagon-like peptide-1(7–36)amide: insulinotropic activities in perfused rat pancreases, and receptor binding and cyclic AMP production in RINm5F cells

Watanabe, Yasuko; Kawai, Koichi; Ohashi, Satoshi; Yokota, Chizuko; Suzuki, Shinichi; Yamashita, K.

doi:10.1677/joe.0.1400045

Cited by 39 publications

(37 citation statements)

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“…Because RINm5F cells have reduced sensitivities to GLP-1 and glucose (26) we used dibutyryl-cAMP as an index of GLP-1 action and membrane depolarization with KCl as an indicator of PKAindependent insulin secretion. The response to 40 mM KCl was similar in all three groups (Fig.…”

Section: Resultsmentioning

confidence: 99%

Anchoring of protein kinase A facilitates hormone-mediated insulin secretion

Lester

Langeberg

Scott

1997

Proc. Natl. Acad. Sci. U.S.A.

181

152

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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...

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Section: Resultsmentioning

confidence: 99%

Anchoring of protein kinase A facilitates hormone-mediated insulin secretion

Lester

Langeberg

Scott

1997

Proc. Natl. Acad. Sci. U.S.A.

181

152

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“…This activity was measured by monitoring the cleavage of a p-nitrophenyl ␣-Dglucoside after 60-min incubation with MLR-1023 or castanospermin (Ridruejo et al, 1989). GLP-1 receptor activation was monitored by measuring cAMP production in RINm5F cells after incubation with MLR-1023 or GLP-1 (5 M) for 30 min (Watanabe et al, 1994). …”

Section: Methodsmentioning

confidence: 99%

MLR-1023 Is a Potent and Selective Allosteric Activator of Lyn Kinase In Vitro That Improves Glucose Tolerance In Vivo

Saporito

Ochman²,

Lipinski³

et al. 2012

J Pharmacol Exp Ther

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2(1H)-pyrimidinone,5-(3-methylphenoxy) (MLR-1023) is a candidate for the treatment of type 2 diabetes. The current studies were aimed at determining the mechanism by which MLR-1023 mediates glycemic control. In these studies, we showed that MLR-1023 reduced blood glucose levels without increasing insulin secretion in vivo. We have further determined that MLR-1023 did not activate peroxisome proliferator-activated ␣, ␦, and ␥ receptors or glucagon-like peptide-1 receptors or inhibit dipeptidyl peptidase-4 or ␣-glucosidase enzyme activity. However, in an in vitro broad kinase screen MLR-1023 activated the nonreceptor-linked Src-related tyrosine kinase Lyn. MLR-1023 increased the V max of Lyn with an EC 50 of 63 nM. This Lyn kinase activation was ATP binding site independent, indicating that MLR-1023 regulated the kinase through an allosteric mechanism. We have established a link between Lyn activation and blood glucose lowering with studies showing that the glucose-lowering effects of MLR-1023 were abolished in Lyn knockout mice, consistent with existing literature linking Lyn kinase and the insulin-signaling pathway. In summary, these studies describe MLR-1023 as a unique blood glucose-lowering agent and show that MLR-1023-mediated blood glucose lowering depends on Lyn kinase activity. These results, coupled with other results (J Pharmacol Exp Ther 342:23-32, 2012), suggest that MLR-1023 and Lyn kinase activation may be a new treatment modality for type 2 diabetes.

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“…GLP-1 acts on receptors located in β-cells in pancreatic islets, thereby causing glucose-dependent insulin secretion [33]. It acts on receptors in the peripheral and central nervous systems, inhibiting secretion of gastric acid, slowing gastric emptying, delaying food absorption, inducing satiety, and reducing appetite [34,35].…”

Section: Discussionmentioning

confidence: 99%