Binding specificity and signal transduction of receptors for glucagon-like peptide-1(7–36)amide and gastric inhibitory polypeptide on RINm5F insulinoma cells

Abstract

Glucagon-like peptide-1(7-36)amide (GLP-1(7-36) amide) and gastric inhibitory polypeptide (GIP), peptides of the glucagon family, stimulate insulin secretion in vitro and in vivo. They possess high N-terminal sequence homology. Binding studies with 125I-labelled GIP and 125I-labelled GLP-1(7-36)amide were performed in RINm5F insulinoma cells to investigate receptor specificity and to compare both receptors directly. Both binding sites were highly ligand-specific: GIP did not bind to the GLP-1(7-36)amide receptor and vice versa. Both peptides increased intracellular cyclic AMP levels; GLP-1(7-36)amide was 100-fold more potent in stimulating cyclic AMP production when compared with GIP. At ranges of 1-10 nmol GLP-1(7-36)amide/l and 0.1-10 nmol GIP/l, corresponding to submaximal binding concentrations, the hormones showed an additive effect on cyclic AMP production. The N-terminal portion of GIP was important for binding, as GIP(1-30) showed almost full binding and biological activity. GIP(17-42) bound in a concentration-dependent manner with approximately 500-fold lower potency than GIP. At concentrations of up to 10 mumol GIP(17-42)/l no stimulation of cyclic AMP was observed.

Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling

“…Both processes can be sensed by the RG-cAMP sensor in the cells. RINm5F cells endogenously express G␣ s -coupled receptors for GLP-1, GIP, and glucagon (27,28). cAMP production in RINm5F cells expressing the RGcAMP sensor showed dose-dependent and saturable responses to GLP-1(7-36) amide, GIP, and glucagon (Fig.…”

“…Cell Culture and Transfection of Cell Lines-The RINm5F cell line is derived from a rat islet cell tumor (25,26) and expresses receptors for GLP-1, GIP, and glucagon (27,28). RINm5F cells were seeded at a density of 3 ϫ 10 5 cells/well in 24-well plates and cultured at 37°C and 5% CO 2 in complete RPMI 1640 medium supplemented with 10% (v/v) FBS, 1 mM sodium pyruvate, 14 mM glucose, 1:1000 penicillin/streptomycin, 2 mM glutamine, and 35 mM sodium bicarbonate, pH 7.4, for 16 h. Before transfection, the medium was removed and replaced with 500 l of fresh complete RPMI 1640.…”

Natriuretic effect by exendin-4, but not the DPP-4 inhibitor alogliptin, is mediated via the GLP-1 receptor and preserved in obese type 2 diabetic mice

“…The GLP-1R is a G protein-coupled receptor, and its activation stimulates the formation of cAMP (14). Systemic application of GLP-1 and EX4 at 1-5 g/kg increased urinary cAMP excretion in rats by 20-fold, and the PKA inhibitor H-89 prevented the inhibitory effect of GLP-1 on bicarbonate reabsorption in renal proximal tubule (6).…”

Isolation of Positive Modulator of Glucagon-like Peptide-1 Signaling from Trigonella foenum-graecum (Fenugreek) Seed

“…To examine the specificity of N55 on GLP-1R signaling, we further analyzed the effect of N55 on cAMP production in RINm5F cells stimulated by the gastric inhibitor polypeptide (GIP). GIP is another incretin, and its receptor is also expressed in RINm5F cells (27). Activation of the GIP receptor leads to production of cAMP in RINm5F cells (19,27).…”

“…GIP is another incretin, and its receptor is also expressed in RINm5F cells (27). Activation of the GIP receptor leads to production of cAMP in RINm5F cells (19,27). The cAMP response to either GIP or exendin 4 (Ex-4, a GLP-1 analog) is not affected by N55 (Fig.…”