exendin-(9-39) and Pancreatic-Neoplasms

We have previously reported severe anorexia abruptly induced in rats 2-3 weeks after they have been transplanted subcutaneously with the glucagonoma MSL-G-AN. Vagotomy did not affect the time of onset and severity of anorexia, and the anorectic state resembles hunger with strongly elevated neuropeptide Y (NPY) mRNA levels in the nucleus arcuatus. We now show that circulating levels of bioactive glucagon-like peptide-1 (GLP-1) (7-36amide) start to increase above control levels exactly at the time of onset of anorexia. At this time-point, bioactive glucagon as well as total glucagon precursors and GLP-1 metabolites are already vastly elevated compared to controls. We further show that intravenous administration of very high concentrations of GLP-1 to hungry schedule-fed rats causes anorexia in a dose-dependent manner, which is blocked by the GLP-1 receptor antagonist exendin (9-39). GLP-1 (7-36amide) has a well-characterized anorectic effect but also causes taste aversion when administered centrally. The anorectic effect is blocked in rats treated neonatally by monosodium glutamate (MSG). We show that MSG treatment does not prevent the MSL-G-AN-induced anorexia, thereby suggesting a different type of anorectic function. We show a very strong component of taste aversion as anorectic rats, when presented to novel or known alternative food items, will resume normal feeding for 1 day, and then redevelop anorexia. We hypothetize that the anorexia in MSL-G-AN tumour-bearing rats correlates with a foetal processing pattern of proglucagon to both glucagon and GLP-1 (7-36amide), and is due to taste aversion. The sudden onset is characterized by a dramatic increase in circulating levels of biologically active GLP-1 (7-36amide), suggesting eventual saturation of proteolytic inactivation of its N-terminus.

Topics: Animals; Anorexia; Appetite Regulation; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagonoma; Male; Neoplasm Transplantation; Pancreatic Neoplasms; Peptide Fragments; Proglucagon; Rats; Receptors, Glucagon; Taste

Radiotracer diagnosis of insulinoma, can be done using somatostatin or glucagon-like peptide 1 (GLP-1). Performance of GLP-1 antagonists tends to be better than of agonists.. We investigated the uptake of the antagonist exendin (9-39), radiolabeled with technetium- 99m. Two different sites of the biomolecule were selected for chelator attachment.. HYNIC-βAla chelator attached to serine (C- terminus) of exendin, was associated with higher tumor uptake than to aspartate (N- terminus).. The chelator position in the biomolecule influenced receptor uptake.

Topics: Animals; Cell Line, Tumor; Chelating Agents; Chromatography, High Pressure Liquid; Glucagon-Like Peptide 1; Insulinoma; Male; Mice; Mice, SCID; Pancreatic Neoplasms; Peptide Fragments; Radionuclide Imaging; Radiopharmaceuticals; Sensitivity and Specificity; Technetium; Tissue Distribution

Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. Moreover, it was recently reported that antagonist tracers were superior to agonist tracers for somatostatin and gastrin-releasing peptide receptor targeting of tumours. The present preclinical study determines therefore the value of an established GLP-1 receptor antagonist for the in vitro visualization of GLP-1 receptor-expressing tissues in mice and humans.. Receptor autoradiography studies with (125)I-GLP-1(7-36)amide agonist or (125)I-Bolton-Hunter-exendin(9-39) antagonist radioligands were performed in mice pancreas and insulinomas as well as in human insulinomas; competition experiments were performed in the presence of increasing concentration of GLP-1(7-36)amide or exendin(9-39).. The antagonist (125)I-Bolton-Hunter-exendin(9-39) labels mouse pancreatic β-cells and mouse insulinomas, but it does not label human pancreatic β-cells and insulinomas. High affinity displacement (IC(50) approximately 2 nM) is observed in mouse β-cells and insulinomas with either the exendin(9-39) antagonist or GLP-1(7-36)amide agonist. For comparison, the agonist (125)I-GLP-1(7-36)amide intensively labels mouse pancreatic β-cells, mouse insulinoma and human insulinomas; high affinity displacement is observed for the GLP-1(7-36)amide in all tissues; however, a 5 and 20 times lower affinity is found for exendin(9-39) in the mouse and human tissues, respectively.. This study reports a species-dependent behaviour of the GLP-1 receptor antagonist exendin(9-39) that can optimally target GLP-1 receptors in mice but not in human tissue. Due to its overly low binding affinity, this antagonist is an inadequate targeting agent for human GLP-1 receptor-expressing tissues, as opposed to the GLP-1 receptor agonist, GLP-1(7-36)amide.

Topics: Animals; Gene Expression Regulation, Neoplastic; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Insulinoma; Isotope Labeling; Mice; Pancreas; Pancreatic Neoplasms; Peptide Fragments; Receptors, Glucagon

Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The present study tested the hypothesis that GLP-1 may modulate insulin receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1 (0.01-100 nM) for different periods (1 min-24 h). Insulin receptor binding was assessed by competitive ligand binding studies. In addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the GLP-1-induced increase in insulin receptor binding. The GLP-1 effect was potentiated by the adenylyl cyclase activator forskolin and the stable cAMP analog Sp-5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3', 5'-monophosphorothioate but was antagonized by the intracellular Ca(2+) chelator 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P<0.05) increased the diminished number of high-capacity/low-affinity insulin binding sites per cell in type 1 diabetics (9,000+/-3,200 vs. 18,500+/-3,600) and in type 2 diabetics (15,700+/-2,100 vs. 28,900+/-1,800) compared with nondiabetic control subjects (25,100+/-2,700 vs. 26,200+/-4,200). Based on our previous experiments in IEC-6 cells and IM-9 lymphoblasts indicating that the low-affinity/high-capacity insulin binding sites may be more specific for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ, Saile B, and Lutz MP. Am J Physiol Endocrinol Metab 276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD. Diabetologia 39: 421-432, 1996), we further investigated the effect of GLP-1 on proinsulin binding in RINm5F cells and monocytes. In both cell types, GLP-1 induced a significant increase in proinsulin binding. We conclude that, in RINm5F cells and in isolated human monocytes, GLP-1 specifically increases the number of high-capacity insulin binding sites that may be functional proinsulin receptors.

Topics: Adult; Animals; Exenatide; Female; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Humans; Insulin; Insulinoma; Male; Monocytes; Pancreatic Neoplasms; Peptide Fragments; Peptides; Proinsulin; Protein Precursors; Rats; Receptors, Glucagon; Tumor Cells, Cultured; Venoms

The observations that glucagon binds to glucagon-like peptide-1 (tGLP-1) receptors have raised the question of whether glucagon receptors mediate the insulinotropic effect of glucagon. We have investigated the presence and selective activation of glucagon and tGLP-1 receptors on tumor-derived cells. Northern blot analysis detected either glucagon or tGLP-1 receptor messenger RNA in hamster (HIT) and mouse (beta TC3) beta-cell lines, respectively, whereas both receptor messenger RNA were revealed in Syrian hamster insulinoma. Their expression in insulinoma plasma membranes was confirmed by specific covalent labeling with either [125I]glucagon or [125I]tGLP-1. Both glucagon and tGLP-1 receptors showed a single class of high affinity binding sites with respective Kd values of 1.11 +/- 0.11 and 0.82 +/- 0.11 nM. [125I]tGLP binding was dose dependently inhibited with a hierarchy of exendin-4 > tGLP-1 > exendin-(9-39) > oxyntomodulin > glucagon. [125I]Glucagon binding was only inhibited by glucagon and oxyntomodulin. Both glucagon and tGLP-1 increased cAMP formation in insulinoma plasma membranes in a dose-dependent manner, with ED50 values of 170.0 +/- 25.0 and 3.1 +/- 0.4 pM, respectively. Exendin-(9-39), a tGLP-1 receptor antagonist, inhibited tGLP-1-induced, but not glucagon-induced, cAMP formation. Our data demonstrate on hamster insulinoma the presence of high affinity glucagon and tGLP-1 receptors selectively coupled to adenylyl cyclase. The observed low affinity of tGLP-1 receptors for glucagon sustains the idea that each hormone has a direct insulinotropic effect.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Cricetinae; Cross-Linking Reagents; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Insulinoma; Islets of Langerhans; Mice; Pancreatic Neoplasms; Peptide Fragments; Peptides; Receptors, Glucagon; RNA, Messenger