Receptors for glucagon-like peptide-1(7–36) amide on rat insulinoma-derived cells

1988 ◽  
Vol 116 (3) ◽  
pp. 357-362 ◽  
Author(s):  
R. Göke ◽  
J. M. Conlon
Keyword(s):  
Specific Binding ◽  
Maximal Response ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Single Class ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Rat Insulinoma

ABSTRACT Specific binding of 125I-labelled glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) to rat insulinoma-derived RINm5F cells was dependent upon time and temperature and was proportional to cell concentration. Binding of radioactivity was inhibited in a concentration-dependent manner by GLP-1(7–36) amide consistent with the presence of a single class of binding site with a dissociation constant (Kd) of 204± 8 pmol/l (mean ± s.e.m.). Binding of the peptide resulted in a dose-dependent increase in cyclic AMP concentrations (half maximal response at 250 ± 20 pmol/l). GLP-1(1–36)amide was approximately 200 times less potent than GLP-1(7–36)amide in inhibiting the binding of 125I-labelled GLP-1(7–36)amide to the cells (Kd of 45±6 nmol/l). Binding sites for GLP-1 (7–36)amide were not present on dispersed enterocytes from porcine small intestine. J. Endocr. (1988) 116, 357–362

Characterization of the receptor for glucagon-like peptide-1(7–36)amide on plasma membranes from rat insulinoma-derived cells by covalent cross-linking

1989 ◽  
Vol 2 (2) ◽  
pp. 93-98 ◽  
Author(s):  
R. Göke ◽  
T. Cole ◽  
J. M. Conlon
Keyword(s):  
Ligand Binding ◽  
Protein Complex ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Binding Protein ◽  
Single Class ◽  
Intact Cells ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Rat Insulinoma

ABSTRACT 125I-Labelled glucagon-like peptide-1(7–36)amide was cross-linked to a specific binding protein in plasma membranes prepared from RINm5F rat insulinoma-derived cells using disuccinimidyl suberate. Consistent with the presence of a single class of binding site on the surface of intact cells, only a single radiolabelled band at Mr 63 000 was identified by SDS-PAGE after solubilization of the ligand—binding protein complex. The band was not observed when 10 nm glucagon-like peptide-1(7–36)amide was included in the binding assay, but 1 μm concentrations of glucagon-like peptide1(1–36)amide, glucagon-like peptide-2 and glucagon did not decrease the intensity of labelling. No change in the mobility of the band was observed under reducing conditions, suggesting that the binding protein in the receptor is not attached to other subunits via disulphide bonds. In control incubations using plasma membranes from pig intestinal epithelial cells, which do not contain specific binding sites for glucagon-like peptide-1(7–36)amide, no cross-linked ligand-binding protein complex was observed.

scholarly journals Ingestion of coffee polyphenols increases postprandial release of the active glucagon-like peptide-1 (GLP-1(7–36)) amide in C57BL/6J mice

2015 ◽  
Vol 4 ◽  
Author(s):  
Yoshie Fujii ◽  
Noriko Osaki ◽  
Tadashi Hase ◽  
Akira Shimotoyodome
Keyword(s):  
Insulin Secretion ◽  
Human Subjects ◽  
Coffee Consumption ◽  
Epidemiological Studies ◽  
Diabetes Risk ◽  
Dependent Manner ◽  
Diabetes In Animals ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Dose Dependent

AbstractThe widespread prevalence of diabetes, caused by impaired insulin secretion and insulin resistance, is now a worldwide health problem. Glucagon-like peptide 1 (GLP-1) is a major intestinal hormone that stimulates glucose-induced insulin secretion from β cells. Prolonged activation of the GLP-1 signal has been shown to attenuate diabetes in animals and human subjects. Therefore, GLP-1 secretagogues are attractive targets for the treatment of diabetes. Recent epidemiological studies have reported that an increase in daily coffee consumption lowers diabetes risk. The present study examined the hypothesis that the reduction in diabetes risk associated with coffee consumption may be mediated by the stimulation of GLP-1 release by coffee polyphenol extract (CPE). GLP-1 secretion by human enteroendocrine NCI-H716 cells was augmented in a dose-dependent manner by the addition of CPE, and was compatible with the increase in observed active GLP-1(7–36) amide levels in the portal blood after administration with CPE alone in mice. CPE increased intracellular cyclic AMP (cAMP) levels in a dose-dependent manner, but this was not mediated by G protein-coupled receptor 119 (GPR119). The oral administration of CPE increased diet (starch and glyceryl trioleate)-induced active GLP-1 secretion and decreased glucose-dependent insulinotropic polypeptide release. Although CPE administration did not affect diet-induced insulin secretion, it decreased postprandial hyperglycaemia, which indicates that higher GLP-1 levels after the ingestion of CPE may improve insulin sensitivity. We conclude that dietary coffee polyphenols augment gut-derived active GLP-1 secretion via the cAMP-dependent pathway, which may contribute to the reduced risk of type 2 diabetes associated with daily coffee consumption.

scholarly journals Refinement of Glucagon-like Peptide 1 Docking to Its Intact Receptor Using Mid-region Photolabile Probes and Molecular Modeling

2011 ◽  
Vol 286 (18) ◽  
pp. 15895-15907 ◽  
Author(s):  
Laurence J. Miller ◽  
Quan Chen ◽  
Polo C.-H. Lam ◽  
Delia I. Pinon ◽  
Patrick M. Sexton ◽  
...  
Keyword(s):  
Family Members ◽  
Receptor Site ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Amino Terminal ◽  
Single Receptor ◽  
Loop Region ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Amino Terminal Domain

The glucagon-like peptide 1 (GLP1) receptor is an important drug target within the B family of G protein-coupled receptors. Its natural agonist ligand, GLP1, has incretin-like actions and the receptor is a recognized target for management of type 2 diabetes mellitus. Despite recent solution of the structure of the amino terminus of the GLP1 receptor and several close family members, the molecular basis for GLP1 binding to and activation of the intact receptor remains unclear. We previously demonstrated molecular approximations between amino- and carboxyl-terminal residues of GLP1 and its receptor. In this work, we study spatial approximations with the mid-region of this peptide to gain insights into the orientation of the intact receptor and the ligand-receptor complex. We have prepared two new photolabile probes incorporating a p-benzoyl-l-phenylalanine into positions 16 and 20 of GLP1(7–36). Both probes bound to the GLP1 receptor specifically and with high affinity. These were each fully efficacious agonists, stimulating cAMP accumulation in receptor-bearing CHO cells in a concentration-dependent manner. Each probe specifically labeled a single receptor site. Protease cleavage and radiochemical sequencing identified receptor residue Leu141 above transmembrane segment one as its site of labeling for the position 16 probe, whereas the position 20 probe labeled receptor residue Trp297 within the second extracellular loop. Establishing ligand residue approximation with this loop region is unique among family members and may help to orient the receptor amino-terminal domain relative to its helical bundle region.

scholarly journals Actions of glucagon-like peptide-1 on KATP channel-dependent and -independent effects of glucose, sulphonylureas and nateglinide

2006 ◽  
Vol 190 (3) ◽  
pp. 889-896 ◽  
Author(s):  
Neville H McClenaghan ◽  
Peter R Flatt ◽  
Andrew J Ball
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Channel Dependent ◽  
Releasing Effect ◽  
Independent Effects

This study examined the effects of glucagon-like peptide-1 (GLP-1) on insulin secretion alone and in combination with sulphonylureas or nateglinide, with particular attention to KATP channel-independent insulin secretion. In depolarised cells, GLP-1 significantly augmented glucose-induced KATP channel-independent insulin secretion in a glucose concentration-dependent manner. GLP-1 similarly augmented the KATP channel-independent insulin-releasing effects of tolbutamide, glibenclamide or nateglinide. Downregulation of protein kinase A (PKA)- or protein kinase C (PKC)-signalling pathways in culture revealed that the KATP channel-independent effects of sulphonylureas or nateglinide were critically dependent upon intact PKA and PKC signalling. In contrast, GLP-1 exhibited a reduced but still significant insulin-releasing effect following PKA and PKC downregulation, indicating that GLP-1 can modulate KATP channel-independent insulin secretion by protein kinase-dependent and -independent mechanisms. The synergistic insulin-releasing effects of combinatorial GLP-1 and sulphonylurea/nateglinide were lost following PKA- or PKC-desensitisation, despite GLP-1 retaining an insulin-releasing effect, demonstrating that GLP-1 can induce insulin release under conditions where sulphonylureas and nateglinide are no longer effective. Our results provide new insights into the mechanisms of action of GLP-1, and further highlight the promise of GLP-1 or similarly acting analogues alone or in combination with sulphonylureas or meglitinide drugs in type 2 diabetes therapy.

Desensitization of the pancreatic β-cell: effects of sustained physiological hyperglycemia and potassium

2006 ◽  
Vol 291 (6) ◽  
pp. E1381-E1387 ◽  
Author(s):  
Hanae Yamazaki ◽  
Kathleen C. Zawalich ◽  
Walter S. Zawalich
Keyword(s):  
Diabetic Patients ◽  
Β Cell ◽  
Physiological Regulation ◽  
Calcium Dependent ◽  
Chronic Hyperglycemia ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
The Impact ◽  
Dose Dependent Increase ◽  
Dose Dependent

The impact of modest but prolonged (3 h) exposure to high physiological glucose concentrations and hyperkalemia on the insulin secretory and phospholipase C (PLC) responses of rat pancreatic islets was determined. In acute studies, glucose (5–20 mM) caused a dose-dependent increase in secretion with maximal release rates 25-fold above basal secretion. When measured after 3 h of exposure to 5–10 mM glucose, subsequent stimulation of islets with 10–20 mM glucose during a dynamic perifusion resulted in dose-dependent decrements in secretion and PLC activation. Acute hyperkalemia (15–30 mM) stimulated calcium-dependent increases in both insulin secretion and PLC activation; however, prolonged hyperkalemia resulted in a biochemical and secretory lesion similar to that induced by sustained modest hyperglycemia. Glucose- (8 mM) desensitized islets retained significant sensitivity to stimulation by either carbachol or glucagon-like peptide-1. These findings emphasize the vulnerability of the β-cell to even moderate sustained hyperglycemia and provide a biochemical rationale for achieving tight glucose control in diabetic patients. They also suggest that PLC activation plays a critically important role in the physiological regulation of glucose-induced secretion and in the desensitization of release that follows chronic hyperglycemia or hyperkalemia.

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

1993 ◽  
Vol 10 (3) ◽  
pp. 259-268 ◽  
Author(s):  
B Gallwitz ◽  
M Witt ◽  
U R Fölsch ◽  
W Creutzfeldt ◽  
W E Schmidt
Keyword(s):  
Cyclic Amp ◽  
Gastric Inhibitory Polypeptide ◽  
Dependent Manner ◽  
Binding Studies ◽  
Concentration Dependent Manner ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
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/1 and 0·1–10 GIP/1, 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 μmol GIP(17–42)/1 no stimulation of cyclic AMP was observed.

scholarly journals A selective small molecule glucagon-like peptide-1 secretagogue acting via depolarization-coupled Ca2+ influx

2009 ◽  
Vol 201 (3) ◽  
pp. 361-367 ◽  
Author(s):  
Jun-ichi Eiki ◽  
Kaori Saeki ◽  
Norihiro Nagano ◽  
Tomoharu Iino ◽  
Mari Yonemoto ◽  
...  
Keyword(s):  
Small Molecule ◽  
Underlying Mechanism ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Compound A ◽  
Fetal Rat ◽  
Small Molecule Compound ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca2+ release from intracellular stores, but to extracellular Ca2+ influx. The response was inhibited by the presence of either L-type Ca2+ channel blockers or K+ ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Gαs- nor Gαq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca2+ influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.

Glucagon-like peptide-1(7–36)amide: characterization of the domain responsible for binding to its receptor on rat insulinoma RINm5F cells

1990 ◽  
Vol 5 (1) ◽  
pp. 33-39 ◽  
Author(s):  
B. Gallwitz ◽  
W. E. Schmidt ◽  
J. M. Conlon ◽  
W. Creutzfeldt
Keyword(s):  
Cyclic Amp ◽  
Rinm5f Cells ◽  
Binding Studies ◽  
Rinm5f Cell ◽  
Radiation Induced ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Dose Dependent ◽  
Rat Insulinoma

ABSTRACT Glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) is a potent stimulator of insulin secretion. Receptors for this hormone have been found on different insulinoma-derived cell lines, e.g. the RINm5F cell line which is derived from a radiation-induced rat insulinoma. To characterize the part of the GLP-1(7–36)amide molecule that is responsible for binding to its receptor on RINm5F cells, binding studies with synthetic C-terminal (GLP-1(21–36)amide) and synthetic N-terminal (GLP-1(7–25)) GLP-1 fragments were carried out. GLP-1(21–36)amide showed dose-dependent binding to the GLP-1(7–36)amide receptor but was approximately 1500 times less potent in inhibiting binding of 125I-labelled GLP-1(7–36)amide than the intact hormone. GLP-1(7–25) at concentrations up to 10 μmol/l did not inhibit binding of label. Neither fragment changed intracellular cyclic AMP concentrations, in contrast to GLP-1(7–36)amide which increased intracellular cyclic AMP. GLP-1(21–36)amide, however, acted as a weak partial antagonist of GLP-1(7–36)amide with respect to GLP-1(7–36)amide-dependent stimulation of cyclic AMP production.

FSH and LH up-regulate epidermal growth factor receptors in rat granulosa cells

1994 ◽  
Vol 140 (2) ◽  
pp. 171-177 ◽  
Author(s):  
H Fujinaga ◽  
M Yamoto ◽  
T Shikone ◽  
R Nakano
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Single Class ◽  
Epidermal Growth ◽  
Dose Dependent

Abstract Epidermal growth factor (EGF) modulates ovarian folliculogenesis and steroidogenesis and its binding sites have been demonstrated in the ovary. We investigated the localization of EGF-binding sites in the rat ovary, and the effects of FSH and LH on EGF binding to cultured granulosa cells. Autoradiographic localization of 125I-labelled mouse EGF-binding sites was demonstrated in the granulosa and luteal cells. Displacement study and Scatchard analysis showed that a single class of specific binding sites for 125I-labelled mouse EGF was present in the granulosa cells, obtained from the ovaries of immature rats treated with diethylstilboesterol. The number of binding sites and the apparent dissociation constant were 4336 binding sites/cell and 3·42 pmol/l respectively. The granulosa cells were cultured for 48 h at 37 °C in medium alone or with increasing amounts of ovine FSH (oFSH; 1–1000 μg/l). FSH treatment increased 125I-labelled mouse EGF binding to the granulosa cells in a dose-dependent manner. After culture with oFSH (100 μg/l) for 48 h, the cells were cultured in medium alone or with increasing amounts of ovine LH (oLH; 1–1000 μg/l) for an additional 48 h. LH treatment also increased 125I-labelled mouse EGF binding in a dose-dependent manner, compared with the control. However, neither FSH nor LH altered receptor-binding affinity. Furthermore, after culture with oFSH (FSH-primed) or oFSH followed by oLH (LH-primed), tissue plasminogen activator (tPA) activities in the conditioned media were examined by fibrin autography. FSH-primed or LH-primed granulosa cells were more responsive to EGF action to induce an increase in tPA activity. In conclusion, it is suggested that functional receptors for EGF in rat granulosa cells are up-regulated by FSH and LH. Journal of Endocrinology (1994) 140, 171–177

Specific binding and degradation of somatostatin by membrane vesicles from pig gut

1986 ◽  
Vol 250 (5) ◽  
pp. G679-G685 ◽  
Author(s):  
M. Weber ◽  
T. Cole ◽  
J. M. Conlon
Keyword(s):  
High Performance ◽  
Specific Binding ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
Concentration Dependent Manner ◽  
Single Class ◽  
Cell Homogenate ◽  
Somatostatin 14

Plasma membrane vesicles were prepared from the basolateral face of pig small intestinal epithelial cells and were enriched in the activity of Na+-K+-ATPase (9-fold relative to the cell homogenate) and ranged in size from 0.15 to 0.40 micron diam. Incubation of somatostatin-14 and [125I-Tyr11]-somatostatin-14 with the vesicles at 37 degrees C resulted in rapid proteolytic degradation of the peptides. Metabolites were isolated by reverse-phase high-performance liquid chromatography and identified by amino acid composition. Cleavages between Ala1-Gly2, Phe6-Phe7, Phe7-Trp8, and Thr10-Phe11 were observed, indicative of aminopeptidase and endopeptidase action. Degradation was inhibited by 1,10-phenanthroline and by bacitracin, and in the presence of these inhibitors and at 21 degrees C binding of [125I-Tyr11]somatostatin-14 to the vesicles was observed. Binding was inhibited in a concentration-dependent manner by somatostatin-14 (half-maximal inhibition at 2.0 +/- 0.1 nM) and by somatostatin-28 (0.8 +/- 0.1 nM) but not by structurally unrelated peptides. The rate of degradation of [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 by basolateral membrane was less than 20 fold that of [125I-Tyr11]somatostatin-14 and a two- to three-fold enhanced binding to the vesicles was observed. Analysis of the inhibition of binding of this analogue by somatostatin-28 indicates the presence of single class of binding site with Kd = 1.3 +/- 0.3 nM. Rapid degradation but no specific binding of somatostatin-14 by brush-border membranes was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

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